Two forms of FRQ, a central component of the Neurospora circadian clock, arise through alternative in-frame initiation of translation. Either form alone suffices for a functional clock at some ...temperatures, but both are always necessary for robust rhythmicity. Temperature regulates the ratio of FRQ forms by favoring different initiation codons at different temperatures; when either initiation codon is eliminated, the temperature range permissive for rhythmicity is demon- strably reduced. This temperature-influenced choice of translation-initiation site represents a novel adaptive mechanism that extends the physiological temperature range over which clocks function. Additionally, a temperature-dependent threshold level of FRQ is required to establish the feedback loop comprising the oscillator. These data may explain how temperature limits permissive for rhythmicity are established, thus providing a molecular understanding for a basic characteristic of circadian clocks.
The circadian oscillator in Neurospora is a negative feedback loop involving as principal players the products of the frequency (frq) locus. frq encodes multiple forms of its protein product FRQ, ...which act to depress the amounts of frq transcript. In this scheme there are two discrete and separable steps to the circadian cycle, negative feedback itself (repression) in which FRQ acts to decrease the levels of its own transcript, and recovery from repression (derepression) in which frq transcript levels return to peak amounts. By introducing an exogenously regulatable frq transgene into a frq loss-of-function strain (frq9), we created an artificial system in which the two separate steps in the circadian cycle can be initiated and followed separately for purposes of observing their kinetics. Under these conditions the frq-FRQ cycle occupies the time scale of a full circadian cycle. During this time, the process of negative feedback of FRQ on frq transcript levels is rapid and efficient; it requires only 3 to 6 h and can be mediated by on the order of 10 molecules of FRQ per nucleus, a level even less than that seen in the normal oscillation. In contrast, recovery from negative feedback requires 14 to 18 h, most of the circadian cycle, during which time de novo FRQ synthesis has stopped, and existing FRQ is progressively posttranslationally modified. Altogether the time required to complete both of these steps is in good agreement with the 22-h observed period length of the normal circadian cycle.
CD40 signaling in B cells and dendritic cells (DCs) is critical for the development of humoral and cell-mediated immunity, respectively. Nuclear factor κB (NF-κB)–inducing kinase (NIK) has been ...implicated as a central transducing kinase in CD40-dependent activation. Here, we show that although NIK is essential for B cell activation, it is dispensable for activation of DCs. Such data provide compelling evidence that different intermediary kinases are used by different cellular lineages to trigger NF-κB activation via CD40.
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