Fig. 2.

Circadian gating of dark-induced gene expression. (A) Day-length dependence of dark-induced profiles. Northern blotting analysis of three representative genes from each group of kai-dependent dark-induced genes (Fig. 1B) under the dark. Type I: pilT, chlB, and dpsA; type II: digA, gap1, and digC; type III: pilH/rre7, clpB, and digE; and type IV: hspA, psb28-2, and digD. When the cells were transferred to the dark after 12 h in the light, the expression profile of each gene was quite different in the WT (black line) and kaiABC-null mutant (red line) strains. When the WT cells were transferred to the dark after 24 h in the light, the expression profiles (green line) changed, becoming essentially similar to those in the mutant strain. (B) Dark-induced profiles of the digB and lrtA genes, which are categorized as kai-independent genes (kai-indep). Data are shown as in panel A. (C) Schematic representation of the experimental schedule. After the cells were entrained in two 12 h/12 h LD cycles, they were placed in the light for the indicated times, ranging from 6 h to 36 h in increments of 6 h, and were then transferred to the dark conditions. (D) Northern blotting analysis of the digA and hspA genes in the dark when they had been transferred after 6 h (L6D) to 36 h (L36D) under LL. The cells were collected at hours 0, 0.5 (30 min), 1, 2, 4, 8, and 12 in the dark. (E) Time of day-dependent induction levels of digA and hspA mRNAs after 4 h in the dark. The mean values and SDs of four independent experiments are shown. These data were initially normalized to the level at hour 0 in the light; the average level after 4 h in the dark under L12D conditions was then deemed to be 1. (F) Densitometric data for the time of day-dependent DigA and HspA protein accumulation in the dark, when the cells had been transferred to the dark after 12 h (L12D) or 24 h (L24D) in the light, analyzed by Western blotting.