been PERFRQT, reflecting the Drosophila period gene Dartmouth Medical School and the Neurospora frequency gene (the fruits of the Hanover, New Hampshire 03755 first decades of genetic and molecular genetic analysis of clocks) and the fact that the Drosophila timeless gene, tim, was still in the process of arriving. This era was Life is a cyclical chemical process that is regulated in spent convincing ourselves that such genes really were four dimensions. We distinguish parts of the cycle: dethe key to understanding how clocks work. Flies and velopment describes the changes from single cell to fungi were PERFRQT systems for working out basic adult, and aging the changes from adult to death. Birth tools, paradigms, and approaches-gene products whose to death, a cycle, and there are cycles within cyclesexpression levels themselves oscillate, the importance circannual rhythms, menstrual cycles, semilunar cycles, of negative feedback, criteria to begin to distinguish and daily 24 hr or circadian cycles. which oscillatory gene products might contribute to the Twice a year we get a reminder of the importance of action of an internal timer as distinct from being output our internal circadian biological clocks. Daylight sav-(reviewed in Dunlap, 1996), and a universal appreciation ings: in October we fall back just an hour, and yet we of the importance of genetics. If overall this left us with wake up an hour early on Monday anyway and think a less than PERFRQT understanding of timing in genmeals are late-but only for a day, until our clocks are eral, at least many found optimism in the sense that we reset. The reminder is about the way we process enviwere, finally, asking the right questions. This naturally ronmental information and time, namely that we use segued into an interlude where light resetting was exexternal time cues (light and temperature changes that plained by two different mechanisms, through transcriptrack the day without) to set an internal clock that guides tional induction of oscillator components in Neurospora the day within. This internal clock is the lens through (Crosthwaite et al., 1995) or protein turnover in Drosophwhich we survey acute external factors; it takes the lead ila (reviewed in Young, 1998). But by mid 1997 the word in determining what we perceive as time. was PASWCCLK (the first clock components with known It used to be that research in chronobiology moved biochemical functions [transcriptional activators], the along at a gentlemanly pace. It was a field in that it first mammalian clock gene, and the first protein domain shared a common set of problems, a common vocabu-[PAS] conserved among clock molecules from different lary, and a series of common assumptions: only eukaryphyla) and then MPERMPER (mammalian orthologs and otes had real clocks and they probably evolved just paralogs of model system clock genes), and then in mid once, since the basic properties of the rhythms were 1998 the already ungainly CYCBMALJRKDBT (and a generally the same. Any cell in fungi, plants, or protists g...

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1, a sequence homolog of Drosophila per, that exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene frq. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

In the fungus Neurospora crassa, the blue light photoreceptor(s) and signaling pathway(s) have not been identified. We examined light signaling by exploiting the light sensitivity of the Neurospora biological clock, specifically the rapid induction by light of the clock component frequency (frq). Light induction of frq is transcriptionally controlled and requires two cis-acting elements (LREs) in the frq promoter. Both LREs are bound by a White Collar-1 (WC-1)/White Collar-2 (WC-2)-containing complex (WCC), and light causes decreased mobility of the WCC bound to the LREs. The use of in vitro-translated WC-1 and WC-2 confirmed that WC-1, with flavin adenine dinucleotide as a cofactor, is the blue light photoreceptor that mediates light input to the circadian system through direct binding (with WC-2) to the frq promoter.

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).The PAS (Per-Arnt-Sim) protein superfamily transduces signals from diverse biological cues, often by coupling cofactor chemistry to alterations in protein conformation or association (1). The canonical PAS domain protein photoactive yellow protein (PYP) and the light, oxygen, or voltage (LOV) PAS subclass sense blue light in bacteria, plants, and fungi (2, 3). Despite extensive photochemical and structural characterization of such bluelight sensors (2, 4-8), the mechanism by which cofactor excitation leads to biological signal propagation remains an open question.The filamentous fungus Neurospora crassa employs two blue-light sensors with LOV domains, White Collar-1 (WC-1) and Vivid (VVD) to regulate a variety of light responses (9). WC-1 and nonphotosensitive WC-2 form a complex (WCC) that resets the circadian clock by activating transcription of the clock oscillator protein Frequency (FRQ), as well as many other genes (9, 10). VVD, a small PAS protein devoid of auxiliary domains, tunes Neurospora's blue-light response by attenuating activation of the WCC. VVD is essential for response to changing levels of light and for adaptation under constant light (11)(12)(13)(14). VVD and WC-1 share sequence similarity in a core LOV domain and surrounding regions (15). Swapping the WC-1 core LOV domain with that from VVD maintains some light responses in Neurospora (16). VVD and WC-1 require flavin adenine dinucleotide (FAD) for activity instead of flavin mononucleotide (FMN), which is used by plant and algal LOV-containing proteins known as phototropins (9,12,17,18).We report the crystal structure of VVD in its dark-and light-adapted states and show how chemical changes at the active center generate conformational change at the N terminus of

The frequency (frq) locus of Neurospora crassa was originally identified in searches for loci encoding components of the circadian clock. The frq gene is now shown to encode a central component in a molecular feedback loop in which the product of frq negatively regulated its own transcript, which resulted in a daily oscillation in the amount of frq transcript. Rhythmic messenger RNA expression was essential for overt rhythmicity in the organism and no amount of constitutive expression rescued normal rhythmicity in frq loss-of-function mutants. Step reductions in the amount of FRQ-encoding transcript set the clock to a specific and predicted phase. These results establish frq as encoding a central component in a circadian oscillator.

We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease