Multiple oscillators regulate circadian gene expression in Neurospora

Abstract

High-density microarrays were used to profile circadian gene expression in Neurospora crassa cultures grown in constant darkness. We identified 145 clock-controlled genes (ccgs). The ccgs peaked in mRNA accumulation at all phases of the day, with the majority peaking in the late night to early morning. The predicted or known functions of the ccgs demonstrate that the clock contributes to a wide range of cellular processes, including cell signaling, development, metabolism, and stress responses. Although the period of rhythm of most of the ccgs was found to depend on the well characterized frequency (FRQ)-based oscillator, three ccgs appeared to have a rhythm that was significantly short in the long period (29-h) frq7 mutant strain. These ccgs accumulate mRNA rhythmically with a circadian period in a frq-null strain, confirming the existence of a second oscillator in N. crassa.

Fig. 1.

(a) Total RNA was harvested from dark-grown clock frq⁺ and frq⁷ cultures after the indicated times in the dark (DD) and probed with ccg-2 to verify rhythmicity of the cultures. The corresponding CT are designated at the top. The microarray tracings of ccg-2 are shown (Right) for both strains. The values (means ± SEMs; n = 4) for each time point are shown. CT (top) and DD (bottom) times are indicated. (b) Circadian clock-regulated genes were grouped into four classes based on their peak time of mRNA accumulation in frq⁺ cells. The values (means ± SEMs) for each time point represent the average of all genes (n) in that class. DD and CT times are indicated below the tracings. (c) Total RNA was isolated from cultures after the indicated times in the dark, and Northern blots were probed with the designated EST clone. rRNA was used as a loading control for each experiment. Relative mRNA levels are plotted as relative band intensity vs. time (below).

Fig. 2.

Microarray tracings of RNA accumulation data from three EST clones in clock frq⁺ and frq⁷ strains. The control ccg-2 tracing is shown for comparison. The values (means ± SEMs; n = 4) for each time point are shown. The times in the dark (DD) are shown below each graph, and the corresponding CT for each strain is indicated at the top.

Fig. 3.

(a) Total RNA was isolated from frq⁺ and frq^- strains harvested after transfer from light to dark (DD) at the indicated times, and Northern blots were probed with the EST clones. rRNA was used to verify equal loading of RNA. Relative mRNA levels are plotted as relative band intensity vs. time in the dark and are shown below the Northern blots for each gene. frq⁺ is shown as a solid line and frq^- as a dashed line. For all four genes, two additional Northern blots using RNA isolated from independent time courses gave similar results (not shown). (b) Mycelia from each culture were assayed on race tubes grown in constant darkness to verify the circadian phenotype. The growth fronts were marked every 24 h (vertical black lines). The centers of the conidial bands are marked with a black dot.