Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific

Abstract

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.

Figure 1

Isolation of RNA from frq⁺ and frq⁷ cultures representing four different times in the circadian day. (A) Densitometry of the clock-regulated ccg-1 mRNA isolated from N. crassa frq⁺ (solid line) and frq⁷ (dotted line) cultures grown in continuous dark and harvested every 4 hr (4). The time in the dark (Hours DD) is indicated on the bottom of the plot, and the corresponding CT is shown on the top for both strains. Arrows point to the time of harvest (DD37 and DD43) used to isolate mRNA for the generation of the time-of-day-specific cDNA libraries. (B) Northern blot hybridization of total RNA (10 μg) extracted from frq⁺ and frq⁷ strains at DD37 and DD43 representing approximate CT18, CT0, CT6, and CT12 as shown in A. Hybridization is to the clock-controlled eas(ccg-2) gene.

Figure 2

Criteria used to establish clock control of gene expression, as illustrated by the ccg-4 gene. The steady-state levels of ccg-4 mRNA were assayed by Northern blot analyses in frq⁺ (A) and frq⁷ (B) strains. Liquid cultures of mycelia were grown in constant darkness and harvested after the indicated times in the dark (Hours DD). The approximate CT at the time of harvests are shown below the autoradiograms. (C) Following autoradiography, ccg-4 mRNA was quantitated by densitometry, and plotted as relative band intensity versus time in the dark for both frq⁺ (solid line) and frq⁷ (dotted line). Equal loading of the RNA was verified by inspection of ethidium bromide stained rRNA on the gel (data not shown)

Figure 3

Rhythmic transcript accumulation of the ccgs in the frq⁷ (29-hr period) strain. RNA was isolated from the frq⁷ mutant strain at the indicated times (Hours DD) representing the approximate CTs shown at the top of the autoradiograms, and hybridized to RNA probes specific for each ccg (shown at left). Equivalent loading of RNA was verified by hybridization to rRNA. Exposure times of the autoradiograms are varied for visualization.

Figure 4

Developmental and light regulation of the ccgs. (A) RNA isolated from tissue 0, 2, 4, and 8 hr after induction of conidiation was hybridized to each new ccg (shown on the left) and to rRNA to verify equivalent loading. (B) RNA was isolated from cultures grown for 22 hr in the dark (CT12) and harvested after an additional 30 min in the dark (D30), or transferred to the light for 30, 60, and 120 min (L30, L60, and L120, respectively). The RNA was hybridized to probes as indicated above.

Figure 5

Clock-controlled genes map throughout the genome. Chromosomal map locations of N. crassa clock-controlled genes, as determined by RFLP analysis, are indicated by arrows on the appropriate linkage groups (this work and ref. 4). Individual linkage groups (LG) are designated with a Roman numeral, followed by left or right in parenthesis, indicating the specific arm of the chromosome represented. The map positions of other conventional markers are shown for reference, and the order of markers denoted in parenthesis are uncertain (37). The centromeres are shown as a circle. The map distances are rough approximations.