Genetic and molecular characterization of a cryptochrome from the filamentous fungus Neurospora crassa.

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.

Principles underlying the complex dynamics of temperature entrainment by a circadian clock.

Autonomously oscillating circadian clocks resonate with daily environmental (zeitgeber) rhythms to organize physiology around the solar day. Although entrainment properties and mechanisms have been studied widely and in great detail for light-dark cycles, entrainment to daily temperature rhythms remains poorly understood despite that they are potent zeitgebers. Here we investigate the entrainment of the chronobiological model organism Neurospora crassa, subject to thermocycles of different periods and fractions of warm versus cold phases, mimicking seasonal variations. Depending on the properties of these thermocycles, regularly entrained rhythms, period-doubling (frequency demultiplication) but also irregular aperiodic behavior occurs. We demonstrate that the complex nonlinear phenomena of experimentally observed entrainment dynamics can be understood by molecular mathematical modeling.

Entrainment elicits period aftereffects in Neurospora crassa.

Circadian clocks continue to oscillate in constant conditions with their own period (tau) and entrain to a cyclic environment by adjusting their intrinsic period to that of the zeitgeber. When circadian clocks are released from entrained to constant conditions, the tau of their initial free-run often depends on the nature of the prior zeitgeber. These postentrainment effects on period (tau-aftereffects) have predominantly been reported for animals but, so far, not fungi. The authors therefore investigated tau aftereffects in the classic circadian model system Neurospora crassa. The standard laboratory strain frq+, the short-period mutant frq(1), and the long-period mutant frq(7) were entrained to 11 different photoperiods in a 24-h day (2-22 h) and to zeitgebers with six different T (16-26 h), and then released to constant darkness. tau-Aftereffects in response to different photoperiods correlated weakly with prior photoperiod in frq+ and were unsystematic in both period mutant strains. Strength and direction of the tau-aftereffect in zeitgeber cycles with different T depended on their length and on the strain, showing a negative correlation with zeitgeber length in frq+ and positive correlations in frq(1) and frq(7). It has been proposed that tau-aftereffects are based on interactions of oscillators within a cellular network. The present findings in Neurospora, which grows as a syncytium, suggest that tau-aftereffects also exist in circadian systems based on multioscillatory networks organized at the molecular level.