A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to acccomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250

Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.

Circadian clocks in fungi and animals are driven by a functionally conserved transcription–translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense-Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.

Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2 , FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the longperiod rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60-120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.