Complex transcriptional control of wc-1. (A) Expression of wc-1 by P_dist and P_prox is required for entrainment. Race tubes were inoculated with the indicated strains and exposed to 12 h:12 h light:dark cycles. Conidial densities were plotted versus time. Phases of conidiation peaks were calculated as zeitgeber time (h; mean±s.e.m.). (B) Regulation of P_dist and P_prox by the WCC. Upper panel: schematic outline of the wc-1 gene. P_dist and P_prox are indicated. A putative CAAT box, TATA box and initiator sequences are shown. The grey boxes (Box 1–3) indicate putative GATN-type binding sites for WCC. Lower panel: sequence comparison of wc-1 (Box 1–3) and the light-responsive elements of frq (Froehlich et al, 2002) and al-3 (Carattoli et al, 1994). The arrows indicate the orientation of GATN elements.

Localization of the light-inducible promoter element of the wc-1 gene. (A) P_prox contains a light-responsive element (LRE). wc-1⁺ (upper panel), wc-1⁺, P_dist-luc and wc-1⁺, P_prox-luc (lower panel) were incubated in constant darkness for 24 h and then transferred to light for the indicated periods. Upper panel: total wc-1 RNA and P_dist-specific RNA were measured by quantitative real-time PCR. Lower panel: luc RNA expressed by the indicated strains was determined by quantitative RT-PCR. RNA levels before light induction were set equal to unity. (B) P_prox transcripts are light inducible. Upper panel: schematic outline showing forward (F) and reverse (R) primers with respect to P_dist and P_prox. Numbers (−907 and −1168) indicate the distance in base pairs from the AUG. Lower panel: semiquantitative PCR (23 cycles) was carried out on complementary DNAs prepared from wc-1⁺ after light exposure for the indicated time periods. Actin was used as a control.

Expression of wc-1 in constant darkness is dependent on the White Collar Complex. (A) WC-1 regulates distinct promoter segments of wc-1 in constant darkness (DD). Indicated constructs (see Fig 2) were integrated into the his3 locus of wc-1⁺ and of Δwc-1. Luciferase (luc) activity was measured in total cell extracts prepared from cultures collected at DD23. 100%, enzyme activity measured in wc-1⁺, P_dist-P_prox-luc. Data are means±s.e.m. of four experiments. (B) wc-1 expression is reduced in the White Collar Complex-deficient strains wc-1^RIP and Δwc-2. Cells were collected at DD24. wc-1 RNA was analysed by quantitative real-time PCR (RT-PCR). Data are means±s.e.m. of three experiments, each carried out in duplicate. (C) Activity of P_dist and P_prox in constant light (LL) and DD. wc-1⁺ was grown in LL (0) and then transferred to DD. Samples were collected at the indicated time points and total wc-1 RNA (solid black line) and P_dist-specific RNA (dotted grey line) were measured by RT-PCR (supplementary information online).

Characterization of the promoter activity of different wc-1 gene segments. (A) Schematic outline of luciferase (luc) reporter constructs. (B) Different wc-1 gene segments exert promoter activity. wc-1⁺,his3 was transformed with the indicated constructs and luc activity was measured in total cell extracts of light-grown cells. Enzyme activity measured in extracts of wc-1⁺, P_dist-P_prox-luc was set to 100%. Data are means±s.e.m. of five to eight experiments. (C) The wc-1.ORF strain expresses an amino-terminally truncated version of WC-1. wc-1⁺ and the strains wc-1.ORF and Δwc-1, qa-wc-1.NΔQ were grown in constant light (LL). Expression of WC-1.NΔQ (amino acids 67–1,167) was induced with quinic acid. Total cell extracts were analysed by SDS–polyacrylamide gel electrophoresis and immunoblotting with anti-WC-1 antibody. (D) Frequency (FRQ) levels in wc-1.ORF are high in LL but reduced in constant darkness (DD). A representative western blot analysis of total cell extract and quantification by densitometry (n=3) is shown. (E) The conidiation rhythm of wc-1.ORF is dampened. Two representative race tubes and the averaged conidial densities are shown.

Complex control of wc-1 transcription. (A) Entrainment of circadian conidiation depends on the wc-1 promoter. Race tubes, inoculated with the indicated strains, were exposed to 1 h:23 h light:dark cycles at 25°C. Averaged conidial densities (n=3) were plotted versus time. (B) Resetting of the clock by light is altered in the Δwc-1, qa-wc-1 (dotted grey line) strain compared with wc-1⁺ (black line). Race tubes were exposed to 20 min light pulses at the indicated time in constant darkness (DD). The phase shift of conidial bands (compared with control strains kept in DD) was blotted versus time when the light pulse was given. Black and dotted bars at the bottom indicate subjective night and day, respectively. (C) The transcription of wc-1 is initiated at various sites. The wc-1 gene is schematically outlined. The positions of four amplicons generated by RNA ligase-mediated rapid amplification of complementary DNA ends are indicated above. The putative promoters P_dist, P_prox and P_int are indicated by arrows.