FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identi®ed Neurospora homologs of casein kinase 1e. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes signi®cantly increased protein stability. A strain harboring the mutant frqDPEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frqDPEST-1 RNA and FRQDPEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components. Keywords: casein kinase 1e/circadian/frequency/ Neurospora/PEST IntroductionMost organisms use circadian clocks to regulate physiological and behavioral activities in a time-of-day speci®c manner. Circadian clocks are cell-autonomous and mediate rhythmic expression of a broad variety of clockcontrolled genes with a periodicity of approximately one day even in the absence of environmental cues. Circadian clocks are entrainable by exogenous stimuli (zeitgebers) such as light, temperature and nutrition. Entrainment synchronizes the endogenous oscillations with the exogenous time and allows behavioral¯exibility. On the molecular level circadian oscillations are supported by interconnected transcriptional±translational feedback loops of considerable complexity. The interconnected loops seem to function in a similar manner in fungi,¯ies and mammals (Dunlap et al., 1999;Young, 2000;Albrecht, 2001;Reppert and Weaver, 2001;Williams and Sehgal, 2001).The circadian system of Drosophila melanogaster is well characterized on the molecular level. Drosophila CLOCK (CLK) and CYCLE (CYC) are Per-Arnt-Sim-like (PAS) domain-containing transcription factors that control transcription of period (per) and timeless (tim) RNA (Allada et al., 1998;Darlington et al., 1998;Rutila et al., 1998;McDonald et al., 2001;Wang et al., 2001). When PER and TIM proteins are synthesized they form a complex that enters the nucleus and represses transcription of their own genes by inactivating CLK±CYC (Hardin et al., 1990;Saez and Young, 1996;Lee et al., 1998Lee et al., , 1999. During the course of a day PER and TIM are degraded, which again allows synthesis of per and tim RNA by CLK±CYC (Young, 2000;Williams and Sehgal, 2001). The length of a circadian period is affected by the kinetics of nuclear entry and degradation of PER±TIM. TIM is phosphorylated by SHAGGY, which regulates nuclear translocation of the PER±TIM heterodimer (Martinek et al., 2001). The casein kinase 1e (CK-1e) homolog DOUBLETIME (DBT) triggers degradation of PER via phosphorylation and thereby regulates period length Rothen¯uh et al....

FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These ®ndings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction.

When deprived of essential nutrients, the non-diazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 undergoes a proteolytic degradation of the phycobiliproteins, its major light-harvesting pigments. This process is known as chlorosis. This paper presents evidence that the degradation of phycobiliproteins is part of an acclimation process in which growing cells differentiate into non-pigmented cells able to endure long periods of starvation. The time course of degradation processes differs for various photosynthetic pigments, for photosystem I and photosystem II activities and is strongly influenced by the illumination and by the experimental conditions of nutrient deprivation. Under standard experimental conditions of combined nitrogen deprivation, three phases of the differentiation process can be defined. The first phase corresponds to the well-known phycobiliprotein degradation, in phase 2 the cells lose chlorophyll a prior to entering phase 3, the fully differentiated state, in which the cells are still able to regenerate pigmentation after the addition of nitrate to the culture. An analysis of the protein synthesis patterns by two-dimensional gel electrophoresis during nitrogen starvation indicates extensive differential gene expression, suggesting the operation of tight regulatory mechanisms.

The nondiazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 responds to nitrogen deprivation by differentiating into nonpigmented resting cells able to survive prolonged periods of starvation. The degradation of photosynthetic pigments, termed chlorosis, proceeds in an ordered manner in which the light-harvesting phycobiliproteins are degraded prior to chlorophyll. Here, we show that the function of the global transcription activator of nitrogen-regulated genes, NtcA, is required for the sequential pigment degradation and cell survival. The P(II) protein, known to signal the nitrogen status of the cells, is most probably not involved in the perception of the nitrogen-starvation-specific signal since in a mutant lacking P(II), chlorosis proceeded in the same manner as in the wild type. Inhibition of glutamine synthetase with l-methionine sulfoximine led to a rapid decrease of apc mRNA and to an increase of nblA mRNA levels, which is characteristic for nitrogen deprivation, suggesting that nitrogen starvation is sensed by a metabolic signal connected to glutamine synthetase activity. However, l-methionine sulfoximine treatment did not induce phycobiliprotein degradation, but led to an immediate cessation of this proteolytic process after its induction by nitrogen deprivation. This suggests that the proteolytic activity elicited by the expression of nblA has to be supported by glutamine synthetase activity.

The filamentous fungus Neurospora crassa is a model organism for the genetic dissection of blue light photoreception and circadian rhythms. WHITE COLLAR-1 (WC-1) and WC-2 are considered necessary for all light responses, while FREQUENCY (FRQ) is required for light-regulated asexual development (conidia formation); without any of the three, self-sustained (circadian) rhythmicity in constant conditions fails. Here we show that light-regulated and self-sustained development occur in the individual or mutant white collar strains. These strains resemble wild type in their organization of the daily bout of light-regulated conidiation. Molecular profiles of light- induced genes indicate that the individual white collar-1 and white collar-2 mutants utilize distinct pathways, despite their similar appearance in all aspects. Titration of fluence rate also demonstrates different light sensitivities between the two strains. The data require the existence of an as-yet-unidentified photoreceptor. Furthermore, the extant circadian clock machinery in these mutant strains supports the notion that the circadian system in Neurospora involves components outside the WC-FRQ loop.