DISSECTION OF A CIRCADIAN OSCILLATION INTO DISCRETE DOMAINS

The circadian oscillator in Neurospora is a negative feedback loop inv olving as principal players the products of the frequency (frq) locus. frq encodes multiple forms of its protein product FRQ, which act to d epress the amounts of frq transcript. In this scheme there are two dis crete and separable steps to the circadian cycle, negative feedback it self (repression) in which FRQ acts to decrease the levels of its own transcript, and recovery from repression (derepression) in which frq t ranscript levels return to peak amounts. By introducing an exogenously regulatable frq transgene into a frq loss-of-function strain (frq(9)) , we created an artificial system in which the two separate steps in t he circadian cycle can be initiated and followed separately for purpos es of observing their kinetics. Under these conditions the frq-FRQ cyc le 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 tha n that seen in the normal oscillation. In contrast, recovery from nega tive feedback requires 14 to 18 h, most of the circadian cycle, during which time de novo FRQ synthesis has stopped, and existing FRQ is pro gressively posttranslationally modified. Altogether the time required to complete both of these steps is in good agreement with the 22-h obs erved period length of the normal circadian cycle.