Analysis of circadian rhythms in Neurospora: overview of assays and genetic and molecular biological manipulation.

The eukaryotic filamentous fungus Neurospora crassa is a tractable model system that has provided numerous insights into the molecular basis of circadian rhythms. In the core circadian clock feedback loop, WC-1 and WC-2 interact via PAS domains to heterodimerize, and this complex acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. In the negative step of the loop, dimers of FRQ feed back to block the activity of the WC-1/WC-2 complex (WCC) and, in a positive step, to promote the synthesis of WC-1. Several kinases phosphorylate FRQ, leading to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light and temperature entrainment of the clock arise from rapid light induction of frq expression and from the effect of elevated temperatures in driving higher levels of FRQ. Noncircadian candidate slave oscillators, termed FRQ-less oscillators (FLOs), have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the core FRQ/WCC feedback loop coordinates the circadian system by regulating downstream clock-controlled genes either directly or via regulation of driven FLOs. This article provides a brief synopsis of the system and describes current assays for the Neurospora clock. Methods for genetic and molecular manipulation of the core clock are summarized, and accompanying chapters address more specifically aspects of photobiology and output.