Molecular mechanism of a FRQ -less oscillator (FLO) in the chol-1 mutant of Neurospora crassa

Neurospora is a model organism for investigating the mechanism of circadian rhythmicity. The FRQ/WCC feedback loop (FWO) is proposed to be the central mechanism of the circadian clock. However, the existence of FRQ-less oscillators (FLOs) challenges this model. The goal of this study is to identify the FLO components in the chol-1 mutant and the FLO's relationship with FWO, which includes four research directions: the regulation of the conidiation rhythm in chol-1 by FWO and FLO; the effects of prd mutations on the FLO; mutagenesis of new clock genes affecting FLO; and cloning and induced over-expression of DAG kinase in Neurospora. .;Clock genes have different effects on the FLO in chol-1 frg10. By crossing prds to chol-1 frg10 double and triple mutants were constructed. It was found that prd-1 and prd2 mutations disrupt stable rhythmicity of chol-1 and abolish the rhythm of chol-1 frq10. prd-3 and prd-4 have less effect on chol-1 and chol-1 frq10 rhythms.;Mutagenesis was used to identify new mutations affecting the FLO. Microconidia from chol-1 frq10 were exposed to UV light and clock related mutations were selected and identified. Long, short and arrhythmic mutants were found. The UV90 mutant was identified as a mutant that has a period of about 21 hr with a slow growth rate. All mutant strains were entrainable to temperature cycles.;A putative DAG kinase gene in Neurospora has been discovered. Its cloning and over-expression were carried out to investigate the relationship between DAG and the clock.;I investigated FRQ protein and frq mRNA rhythms and conidiation rhythms in chol-1. chol-1 has 24 hr FRQ and frq mRNA rhythms, however, chol-1 with choline has a conidiation period of 24 hr, while chol-1 without choline has a long conidiation period about 43 hr. Temperature combined with light entrainment can entrain the conidiation bands to 48 hrs. FRQ protein degradation rates are the same in chol+ and chol-1. Band ending assay indicates that short period (24 hr) molecular rhythms are synchronized with the long period conidiation rhythm. The conidiation in chol-1 is gated therefore conidiation bands do not appear every 24 hr. A FLO may be gating the conidiation banding.;Collectively, this study has revealed that chol-1 has a 24 hr FRQ and frq mRNA rhythm while it has a long conidiation rhythm. The conidiation in chol-1 is gated therefore conidiation bands do not appear every 24 hr rather than about 43 hr. prd-1, prd2 and UV90 are found to be possible components for FLO in conidiation.;We use Neurospora as a model organism for its excellent genetics and sequenced genome as well as rhythmic visible conidiation. Our research on the FWO and FLO relationship in chol-1 provides an important foundation for understanding the role of multiple oscillators in the circadian system and the links between the clock and metabolism.