Identification and initial characterization of circadian clock mutants in Neurospora crassa

Abstract

Circadian clocks in organisms provide a means to coordinate behavioral, physiological, and biochemical activities to the appropriate time of day. For example, in Neurospora crassa, the circadian clock controls asexual spore formation (conidiation). In constant darkness, conidiation occurs every 22 hrs and this rhythm can be easily viewed on a specialized culture tube called a race tube. Using the race tube assay to screen for mutations in genes that alter the conidiation rhythm, a few central clock components have been identified. These include the frequency gene, the white collar-1 and white collar-2 genes, and the period genes. While this approach was fruitful, the screens were not saturating and many genes were likely missed. Furthermore, no signaling components involved in the conveyance of time information from the clock to the overt rhythms (the output pathways) are known in N. crassa. To identify additional clock-associated genes in N. crassa, a novel genetic selection has been developed. This selection involved the fusion of the promoter region of a clock-controlled gene to a selectable marker. Following reconstruction experiments, the selection has been used to identify mutations that affect expression of the chimeric gene. Preliminary characterization of the mutations reveals one class of mutants that are rhythmic in the absence of frequency. These findings support a model that is similar to clocks in higher eukaryotes, in which the N. crassa clock is built using a multi-oscillator system.