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mutation of msh-2 in neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence.the neurospora homologue msh-2 of the escherichia coli mismatch repair gene muts was mutated by repeat-induced point mutation (rip) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(rip-lk1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele k458. in msh-2(rip-lk1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. ...200717475521
characterization of alcohol dehydrogenase 1 and 3 from neurospora crassa fgsc2489.alcohol dehydrogenase (adh) is a key enzyme in the production and utilization of alcohols. some also catalyze the formation of carboxylate esters from alcohols and aldehydes. the adh1 and adh3 genes of neurospora crassa fgsc2489 were cloned and expressed in recombinant escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. homology analysis and sequence alignment of amino acid sequence indicated that adh1 and adh3 of n. crassa contained a zinc-bi ...200717516063
biochemical characterization of an l-xylulose reductase from neurospora crassa.an l-xylulose reductase identified from the genome sequence of the filamentous fungus neurospora crassa was heterologously expressed in escherichia coli as a his(6) tag fusion protein, purified, and characterized. the enzyme may be used in the production of xylitol from the major pentose components of hemicellulosic waste, d-xylose and l-arabinose.200717261518
catalase-1 (cat-1) and nucleoside diphosphate kinase-1 (ndk-1) play an important role in protecting conidial viability under light stress in neurospora crassa.recently we reported that catalase-1 (cat-1) played an important role in protecting conidial viability in neurospora crassa, and interacted with a light signal transducer, nucleoside diphosphate kinase-1 (ndk-1). to disclose the functional interaction between cat-1 and ndk-1 at the genetic level, we created cat-1 and ndk-1 double mutants, cat-1;ndk-1-1 and cat-1;ndk-1-2, by crossing single mutants of cat-1 ( rip ) and ndk-1 ( p72h ) previously isolated in our laboratory. the double mutant strain ...200717636331
evidence that rna silencing functions as an antiviral defense mechanism in fungi.the role of rna silencing as an antiviral defense mechanism in fungi was examined by testing the effect of dicer gene disruptions on mycovirus infection of the chestnut blight fungus cryphonectria parasitica. c. parasitica dicer-like genes dcl-1 and dcl-2 were cloned and shown to share a high level of predicted amino acid sequence identity with the corresponding dicer-like genes from neurospora crassa [ncdcl-1 (50.5%); ncdcl-2 (38.0%)] and magnaporthe oryzae [mdl-1 (45.6%); mdl-2 (38.0%)], respe ...200717646660
purification and antimicrobial activity studies of the n-terminal fragment of ubiquitin from human amniotic fluid.a 4.3-kda antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and c18 reversed-phase high performance liquid chromatography. this peptide, which we named amniotic fluid peptide-1 (afp-1), possessed antimicrobial activity but lacked hemolytic activity. in addition, afp-1 potently inhibited the growth of a variety of bacteria (escherichia coli, salmonella typhimurium, listeria monocytogenes and staphylococcus aureus), filamentous fungi (botrytis cinerea, aspe ...200717669700
molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus thermomyces lanuginosus.based on the conserved amino acid sequence (dlkpen) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. total rna was isolated from the thermophilic fungus thermomyces lanuginosus. using race-pcr, full-length cdna of a putative serine-threonine protein kinase gene was cloned from t. lanuginosus. the full-length cdna of t. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precu ...200717676472
neurosprora crassa rad5 homologue, mus-41, inactivation results in higher sensitivity to mutagens but has little effect on pcna-ubiquitylation in response to uv-irradiation.the dna replication machinery stalls at damaged sites on dna. postreplicaton repair (prr) is a system to avoid cell death in such circumstances of deadlock. in saccharomyces cerevisiae, the rad6/rad18 heterodimer plays pivotal roles in prr. it promotes translesion synthesis via the monoubiquitylation of the dna sliding clamp, pcna. ubc13/mms2/rad5 can extend the ubiquitin chain from this monoubiquitylated pcna with a non-canonical lysine 63-linked ubiquitin-chain, resulting in an error-free mode ...200717703305
domain architectures of the scm3p protein provide insights into centromere function and evolution.recently, scm3p has been shown to be a nonhistone component of centromeric chromatin that binds stoichiometrically to cenh3-h4 histones, and to be required for the assembly of kinetochores in saccharomyces cerevisiae. scm3p is conserved across fungi, and displays a remarkable variation in protein size, ranging from approximately 200 amino acids in s. cerevisiae to approximately 1300 amino acids in neurospora crassa. this is primarily due a variable c-terminal segment that is linked to a conserve ...200717704645
sequence analysis and expression of a blue-light photoreceptor gene, le.phra from the basidiomycetous mushroom lentinula edodes.we cloned and sequenced a photoreceptor gene (le.phra) from the basidiomycete lentinula edodes. the product of le.phra, le.phra (924 aa residues) contained a serine-rich region, an lov domain and two pas domains. it was clearly smaller than other fungal lov domain-containing blue-light photoreceptors such as coprinopsis cinerea dst1 (1,175 aa), neurospora crassa wc-1 (1,167 aa), and cryptococcus neoformans wc-1 (1,141 aa). the le.phra gene was found to be transcribed at all stages of the fruitin ...200717827679
comparison of protein coding gene contents of the fungal phyla pezizomycotina and saccharomycotina.several dozen fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. in addition comparative genomics has been used to analyse specific sub groups of fungi. notably, analysis of the phylum saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. however, little has been done to gain a comprehen ...200717868481
involvement of os-2 map kinase in regulation of the large-subunit catalases cat-1 and cat-3 in neurospora crassa.neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases cat-1 and cat-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes cat-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by os-2 map kinase (noguchi et al., fungal genet. biol. 44, 208-218), encodes a small-subunit catalase with unknown function. to clarify the contribut ...200717895581
the use of fungal in vitro systems for studying translational regulation.the use of cell-free systems enables biochemical determination of factors and mechanisms contributing to translational processes. the preparation and use of cell-free translation systems from the fungi saccharomyces cerevisiae and neurospora crassa are described. examples provided illustrate the use of these systems, in conjunction with luciferase assays, [(35)s]met incorporation, and primer-extension inhibition (toeprint) analyses, to assess the translational effects of upstream open reading fr ...200717913625
identification and characterization of amidase- homologous ami1 genes of bottom-fermenting yeast.it has been proposed that a bottom-fermenting yeast strain of saccharomyces pastorianus is a natural hybrid between s. cerevisiae and s. bayanus and possesses at least two types of genome. in the process of conducting expressed sequence tag (est) analysis, we isolated bottom-fermenting yeast-specific (bfy) genes that have no significant homology with sequences in the s288c database. one of the bfy genes, ami1, encodes a protein with homology to an amidase conserved among plants, bacillus subtili ...200717924455
studies on fungal pumilio gene family through mining multiple genome-scale data sets.the genes belonging to pumilio gene family of the fission yeast schizosaccharomyces pombe (s. pombe) were compared with genome information of the several fungi and their functions have been inferred using public-access databases disclosed on the internet websites. the pumilio-family genes are conserved from yeast to man and have been considered to suppress the expression of other genes by binding their specific target messenger rnas. in s. pombe genome, nine genes belonging to pumilio gene famil ...200717932456
cloning, characterization, and mutational analysis of a highly active and stable l-arabinitol 4-dehydrogenase from neurospora crassa.an nad(+)-dependent l-arabinitol 4-dehydrogenase (lad, ec 1.1.1.12) from neurospora crassa was cloned and expressed in escherichia coli and purified to homogeneity. the enzyme was a homotetramer and contained two zn(2+) ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (sdhs). high enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. the enzyme showed strong prefe ...200717938906
the mitochondrial machinery for import of precursor proteins.mitochondria contain a small genome that codes for approx 1% of the total number of proteins that reside in the mitochondria. hence, most mitochondrial proteins are encoded for by the nuclear genome. after transcription in the nucleus these proteins are synthesized by cytosolic ribosomes. like proteins destined for other organellar compartments, mitochondrially destined proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assis ...200717951683
protein targeting to mitochondria of saccharomyces cerevisiae and neurospora crassa: in vitro and in vivo studies.most studies on the biogenesis of mitochondrial proteins have been carried out using fungal mitochondria as a model system. in particular, baker's yeast, saccharomyces cerevisiae, combines several experimental advantages, allowing both genetic and biochemical approaches and thus a combination of investigations in vivo and in vitro. however, the red bread mold neurospora crassa has also been an important research tool. isolated mitochondria can be used from both organisms for import experiments i ...200717951686
improved protocols for functional analysis in the pathogenic fungus aspergillus flavus.an available whole genome sequence for aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. large-scale functional analysis requires an efficient genetic transformation system and the ability to ...200718039373
neurospora crassa as a model organism for mitochondrial biogenesis.neurospora crassa has proven to be an excellent organism for studying various aspects of the biology of mitochondria by biochemical and genetic approaches. as n. crassa is an obligate aerobe and contains complex i, its mitochondria are more similar to mammalian mitochondria than those of yeast. the recent sequencing of the genome of n. crassa and a gene knockout project that is under way make the organism even more attractive. we describe some of the advantages of n. crassa as a model organism a ...200718314721
[homology-dependent inactivation of ltr retrotransposons in genomes of aspergillus fumigatus and a. nidulans].repeat-induced point mutation (rip) is the most intriguing among the known mechanisms of repeated sequences inactivation because of its ability to produce irreversible mutation of repeated dna. discovered for the first time in neurospora crassa, rip is characterized by c:g to t:a transitions in duplicated sequences. the mechanisms and distribution of rip are still purely investigated. mobile elements are a common target for the processes which lead to homology-dependent silencing because of thei ...200718318114
fungal photoreceptors: sensory molecules for fungal development and behaviour.light regulates fungal development and behaviour and activates metabolic pathways. in addition, light is one of the many signals that fungi use to perceive and interact with the environment. in the ascomycete neurospora crassa blue light is perceived by the white collar (wc) complex, a protein complex formed by wc-1 and wc-2. wc-1 is a protein with a flavin-binding domain and a zinc-finger domain, and interacts with wc-2, another zinc-finger domain protein. the wc complex operates as a photorece ...200717609765
crystal structure of human mitochondrial tyrosyl-trna synthetase reveals common and idiosyncratic features.we report the structure of a strictly mitochondrial human synthetase, namely tyrosyl-trna synthetase (mt-tyrrs), in complex with an adenylate analog at 2.2 a resolution. the structure is that of an active enzyme deprived of the c-terminal s4-like domain and resembles eubacterial tyrrss with a canonical tyrosine-binding pocket and adenylate-binding residues typical of class i synthetases. two bulges at the enzyme surface, not seen in eubacterial tyrrss, correspond to conserved sequences in mt-tyr ...200717997975
biological rhythms workshop ia: molecular basis of rhythms generation.current circadian models are based on genetic, biochemical, and structural data that, when combined, provide a comprehensive picture of the molecular basis for rhythms generation. these models describe three basic elements-input pathways, oscillator, and output pathways-to which each molecular component is assigned. the lines between these elements are often blurred because some proteins function in more than one element of the circadian system. the end result of these molecular oscillations is ...200718419259
mitochondrial dna sequences in the nuclear genome of a locust.the endosymbiotic theory of the origin of mitochondria is widely accepted, and implies that loss of genes from the mitochondria to the nucleus of eukaryotic cells has occurred over evolutionary time. however, evidence at the dna sequence level for gene transfer between these organelles has so far been limited to a single example, the demonstration that a mitochondrial atpase subunit gene of neurospora crassa has an homologous partner in the nuclear genome. from a gene library of the insect, locu ...20076298629
[detection in neurospora crassa of a new enzyme--1,3-diphosphoglycerate: polyphosphate phosphotransferase]. 20075557827
[determination of the degree of polymerization of different fractions of inorganic polyphosphates in the mycelium of neurospora crassa (28610a)]. 20075556032
dominance and complementation relationships of conidial longevity mutants of neurospora crassa.previously we reported the occurrence of 16 linked conidial longevity determinant genes in neurospora. mutations of those genes are characterized by a reduction of longevity, a pleiotropic morphological defect, and deficiency of five antioxygenic enzymes. on the basis of the linkage and biochemical data, it was proposed that the genes are spatially and functionally redundant. the results of the present investigation support the hypothesis of functional redundancy. all of the mutants examined wer ...20076233463
what determines growth direction in fungal hyphae?we used high-resolution video microscopy and image analysis to map the trajectory of the spitzenkörper in growing hyphae of neurospora crassa and to correlate it with growth directionality. the spitzenkörper followed a tortuous trajectory produced by a dominant forward motion accompanied by frequent, transverse oscillations. in hyphae with a fixed growth direction, the regression line of the spitzenkörper trajectory corresponded to the longitudinal axis of the hypha. a permanent change in growth ...20079742196
the ww domain protein pro40 is required for fungal fertility and associates with woronin bodies.fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. however, the molecular determinants regulating this cellular process are largely unknown. here we show that the sterile pro40 mutant is defective in a 120-kda ww domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete sordaria macrospora. although ww domains occur in many eukaryotic proteins, homol ...200717351077
neurospora spore killers sk-2 and sk-3 suppress meiotic silencing by unpaired dna.in neurospora crassa, pairing of homologous dna segments is monitored during meiotic prophase i. any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired dna (msud). two genes required for msud have been described previously: sad-1 (suppressor of ascus dominance), encoding an rna-directed rna polymerase, and sad-2, encoding a protein that controls the perinuclear localization of sa ...200717339226
kinetics of circadian band development in neurospora crassa.the circadian rhythm of neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. to follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. the circadian behavior of n. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultiv ...200717329132
mutagenic analysis of an invariant aspartate residue in chorismate synthase supports its role as an active site base.chorismate synthase catalyzes the anti-1,4-elimination of the 3-phosphate and the c(6pror) hydrogen from 5-enolpyruvylshikimate 3-phosphate (epsp) to generate chorismate, the final product of the common shikimate pathway and a precursor for the biosynthesis of aromatic compounds. the enzyme has an absolute requirement for reduced fmn, which is thought to facilitate cleavage of c-o bonds by transiently donating an electron to the substrate. the crystal structure of the enzyme revealed that epsp i ...200717326665
cyclosporin a-resistance based gene placement system for neurospora crassa.dna introduced into neurospora crassa are usually inserted at random ectopic sites of the genome, often in multiple copies. to facilitate the study of gene expression and function, transformation by a single-copy of a gene at a defined locus is desired. although several targeted gene placement methods are available for n. crassa, they all require a specific genetic background in the recipient. we describe here the development of a new locus for targeted gene placement that does not require any p ...200717320431
a genetic network for the clock of neurospora crassa.a diverse array of organisms from bacteria to humans may have evolved the ability to tell time in the presence or absence of external environmental cues. in the lowly bread mould, neurospora crassa, biomolecular reactions involving the white-collar-1 (wc-1), white-collar-2 (wc-2), and frequency (frq) genes and their products constitute building blocks of a biological clock. here we use genetic network models to explain quantitatively, from a systems perspective, how these building blocks interac ...200717301235
role of the conserved cysteine residues of the 11.5 kda subunit in complex i catalytic properties.mitochondrial complex i exists as a mixture of two inter-convertible forms: active (a) and de-activated (d), the latter being sensitive to sh-modifying compounds. to investigate if the conserved cysteine-rich 11.5 kda subunit of neurospora crassa complex i is involved in this process, we subjected the corresponding genomic dna to site-directed mutagenesis. the four cysteine residues of the subunit were separately substituted with serine residues and the resulting proteins were independently expr ...200717261544
circadian rhythms in neurospora crassa: clock mutant effects in the absence of a frq-based oscillator.in neurospora, the circadian rhythm is expressed as rhythmic conidiation driven by a feedback loop involving the protein products of frq (frequency), wc-1 (white collar-1), and wc-2, known as the frq/wc (fwc) oscillator. although strains carrying null mutations such as frq(10) or wc-2delta lack a functional fwc oscillator and do not show a rhythm under most conditions, a rhythm can be observed in them by the addition of geraniol or farnesol to the media. employing this altered media as an assay, ...200717237512
identification of an alternative oxidase induction motif in the promoter region of the aod-1 gene in neurospora crassa.the nuclear aod-1 gene of neurospora crassa encodes the alternative oxidase and is induced when the standard cytochrome-mediated respiratory chain of mitochondria is inhibited. to study elements of the pathway responsible for alternative oxidase induction, we generated a series of mutations in the region upstream from the aod-1 structural gene and transformed the constructs into an aod-1 mutant strain. transformed conidia were plated on media containing antimycin a, which inhibits the cytochrome ...200717237510
identification of genes displaying differential expression in the nuc-2 mutant strain of the mold neurospora crassa grown under phosphate starvation.subtractive hybridization was used to isolate transcripts up-regulated in the nuc-2 mutant strain of neurospora crassa grown under phosphate starvation. following differential screening, 66 cdna clones of the total enriched were screened in a second round by reverse northern hybridization. the 17 cdna candidates displaying visual positive differential expression were sequenced, and functional grouping identified putative proteins possibly involved in diverse cellular processes as, for example, p ...200717229059
hydrophobin genes and their expression in conidial and aconidial neurospora species.homologs of the gene encoding the hydrophobin eas from neurospora crassa have been identified both in the other conidial species of neurospora (n. discreta, n. intermedia, n. sitophila, and n. tetrasperma) and selected aconidial species (n. africana, n. dodgei, n. lineolata, n. pannonica, and n. terricola). southern blot analysis indicated the presence of a single gene in all species examined. eas-like proteins were purified from the conidial species and each was shown to be the proteolytically ...200717218129
circadian rhythmicity during prolonged chemostat cultivation of neurospora crassa.following exposure to light and attainment of steady-state in the chemostat, neurospora was grown in constant conditions of darkness at 25 degrees c for 6 days. biomass samples were taken every 4h for the extraction of rna and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mrnas; frequency (frq), antisense frq (qrf) and clock-controlled gene-14 (ccg-14), and by monitoring the clock-controlled rhythm of sporulation. our results in ...200717196855
the use of amphipathic polymers for cryo electron microscopy of nadh:ubiquinone oxidoreductase (complex i).in the three-dimensional (3d) structure determination of macromolecules, cryo electron microscopy (cryo-em) is an important method for obtaining micrographs of unstained specimens for the single-particle reconstruction approach. for cryo-em, proteins are fixed in a frozen hydrated state by quick-freezing in a thin water layer on a holey carbon film. cryo-em of detergent-solubilized membrane proteins is hindered by the fact that detergents reduce the surface tension of water, so that it is diffic ...200717760617
simple sequence repeats provide a substrate for phenotypic variation in the neurospora crassa circadian clock.white collar-1 (wc-1) mediates interactions between the circadian clock and the environment by acting as both a core clock component and as a blue light photoreceptor in neurospora crassa. loss of the amino-terminal polyglutamine (npolyq) domain in wc-1 results in an arrhythmic circadian clock; this data is consistent with this simple sequence repeat (ssr) being essential for clock function.200717726525
the fungal opsin gene nop-1 is negatively-regulated by a component of the blue light sensing pathway and influences conidiation-specific gene expression in neurospora crassa.we previously demonstrated that the nop-1 gene encodes a putative green-light opsin photoreceptor that is highly expressed in cultures that support asexual sporulation (conidiation) in neurospora crassa. in this study, we demonstrate that nop-1 is a late-stage conidiation gene, through analysis of nop-1 transcript levels in wild-type strains and mutants blocked at various stages of conidiation. nop-1 message amounts are similar with constant illumination or darkness during conidiation, consisten ...200717676324
the pas/lov protein vivid controls temperature compensation of circadian clock phase and development in neurospora crassa.circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. to allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events. here we show that the neurospora protein vivid (vvd) buffers the circadian system against ...200717671094
alternative splicing gives rise to different isoforms of the neurospora crassa tob55 protein that vary in their ability to insert beta-barrel proteins into the outer mitochondrial membrane.tob55 is the major component of the tob complex, which is found in the outer membrane of mitochondria. a sheltered knockout of the tob55 gene was developed in neurospora crassa. when grown under conditions that reduce the levels of the tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import beta-barrel proteins. surprisingly, western blots of wild-type mitochondrial proteins revealed two bands for tob55 that d ...200717660559
high-density detection of restriction-site-associated dna markers for rapid mapping of mutated loci in neurospora.the wealth of sequence information available for neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. although "reverse" genetics, in which genes are first identified by their sequence rather than by their mutant phenotypes, serves as a valuable new approach for elucidating biological processes, classical "forward" genetic analysis is still extremely useful. unfortunately, mapping mutations and identifying the corresponding genes has typica ...200717660537
spitzenkorper localization and intracellular traffic of green fluorescent protein-labeled chs-3 and chs-6 chitin synthases in living hyphae of neurospora crassa.the subcellular location and traffic of two selected chitin synthases (chs) from neurospora crassa, chs-3 and chs-6, labeled with green fluorescent protein (gfp), were studied by high-resolution confocal laser scanning microscopy. while we found some differences in the overall distribution patterns and appearances of chs-3-gfp and chs-6-gfp, most features were similar and were observed consistently. at the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the ...200717644657
genome-wide expression analysis of genetic networks in neurospora crassa.the products of five structural genes and two regulatory genes of the qa gene cluster of neurospora crassa control the metabolism of quinic acid (qa) as a carbon source. a detailed genetic network model of this metabolic process has been reported. this investigation is designed to expand the current model of the qa reaction network. the ensemble method of network identification was used to model rna profiling data on the qa gene cluster. through microarray and cluster analysis, genome-wide ident ...200717597928
design and analysis of an efficient recursive linking algorithm for constructing likelihood based genetic maps for a large number of markers.a multi-locus likelihood of a genetic map is computed based on a mathematical model of chromatid exchange in meiosis that accounts for any type of bivalent configuration in a genetic interval in any specified order of genetic markers. the computational problem is to calculate the likelihood (l) and maximize l by choosing an ordering of genetic markers on the map and the recombination distances between markers. this maximum likelihood estimate (mle) could be found either with a straightforward al ...200717589960
the band mutation in neurospora crassa is a dominant allele of ras-1 implicating ras signaling in circadian output.band, an allele enabling clear visualization of circadianly regulated spore formation (conidial banding), has remained an integral tool in the study of circadian rhythms for 40 years. bd was mapped using single-nucleotide polymorphisms (snps), cloned, and determined to be a t79i point mutation in ras-1. alterations in light-regulated gene expression in the ras-1(bd) mutant suggests that the neurospora photoreceptor white collar-1 is a target of ras signaling, and increases in transcription of bo ...200717575051
conformational switching in the fungal light sensor vivid.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 flavi ...200717510367
model of hyphal tip growth involving microtubule-based transport.we propose a simple model for mass transport within a fungal hypha and its subsequent growth. inspired by the role of microtubule-transported vesicles, we embody the internal dynamics of mass inside a hypha with mutually excluding particles progressing stochastically along a growing one-dimensional lattice. the connection between long-range transport of materials for growth and the resulting extension of the hyphal tip has not previously been addressed in the modeling literature to our knowledge ...200717500728
localization of rho-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus neurospora crassa.rho-4 mutants of the filamentous fungus neurospora crassa lack septa and asexual spores (conidia) and grow slowly. in this report, localization of green fluorescent protein-tagged rho-4 is used to elucidate the differences in factors controlling rho-4 localization during vegetative growth versus asexual development. rho-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; rho-4 persists around the septal pore after septum ...200717496127
fkbp22 is part of chaperone/folding catalyst complexes in the endoplasmic reticulum of neurospora crassa.fkbp22 is a dimeric protein in the lumen of the endoplasmic reticulum, which exhibits a chaperone as well as a ppiase activity. it binds via its fk506 binding protein (fkbp) domain directly to the hsp70 chaperone bip that stimulates the chaperone activity of fkbp22. here we demonstrate additionally the association of fkbp22 with the molecular chaperones and folding catalysts grp170, alpha-subunit of glucosidase ii, pdi, erp38, and cyp23. these proteins are associated with fkbp22 in at least two ...200717470367
control of alternative rna splicing and gene expression by eukaryotic riboswitches.bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. the most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (tpp), which is a derivative of vitamin b1. representatives of this class have also been identified in fungi and plants, where they are predicted to control messenger rna splicing or processing. we examined ...200717468745
turgor regulation in the osmosensitive cut mutant of neurospora crassa.the internal hydrostatic pressure (turgor) of fungal cells is maintained at 400-500 kpa. the turgor is regulated by changes in ion flux and by production of the osmotically active metabolite glycerol. in neurospora crassa, there are at least two genetically distinct pathways that function in adaptation to hyperosmotic shock. one involves a mitogen-activated protein (map) kinase cascade (kinases os-4, os-5 and os-2 downstream of the osmosensing os-1); the other is less understood, but involves th ...200717464067
the peroxin pex14 of neurospora crassa is essential for the biogenesis of both glyoxysomes and woronin bodies.in the filamentous fungus neurospora crassa, glyoxysomes and woronin bodies coexist in the same cell. because several glyoxysomal matrix proteins and also hex1, the dominant protein of woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. here we analyzed the function of the neurospora ortholog of pex14, an essential component of the peroxisomal import machinery. pex14 interac ...200717461798
the neurospora crassa circadian clock.the filamentous fungus neurospora crassa is one of a handful of model organisms that has proven tractable for dissecting the molecular basis of a eukaryotic circadian clock. work on neurospora and other eukaryotic and prokaryotic organisms has revealed that a limited set of clock genes and clock proteins are required for generating robust circadian rhythmicity. this molecular clockwork is tuned to the daily rhythms in the environment via light- and temperature-sensitive pathways that adjust its ...200717452245
neurospora crassa fkbp22 is a novel er chaperone and functionally cooperates with bip.fk506 binding proteins (fkbps) belong to the family of peptidyl prolyl cis-trans isomerases (ppiases) catalyzing the cis/trans isomerisation of xaa-pro bonds in oligopeptides and proteins. fkbps are involved in folding, assembly and trafficking of proteins. however, only limited knowledge is available about the roles of fkbps in the endoplasmic reticulum (er) and their interaction with other proteins. here we show the er located neurospora crassa fkbp22 to be a dimeric protein with ppiase and a ...200717428499
in vitro phosphorylation and kinase assays in neurospora crassa.phosphorylation assay is a widespread technique usually necessary for the identification of a specific kinase substrate and/or for the measurement of kinase activity. as an example of the technique, here we describe an assay aimed to test the phosphorylation of the myelin basic protein (mbp) by protein kinase c (pkc), which is overexpressed and purified from neurospora. the kinase is immunopurified from neurospora using the expression vector pmyx2 and the flag epitope. the purified pkc and the m ...200717417029
northern analysis of sense and antisense frequency rna in neurospora crassa.in northern analysis the presence of specific rna transcripts is detected and their quantity can be estimated. rna is separated using denaturing agarose gel electrophoresis and is subsequently transferred and fixed to a solid support, such as a nitrocellulose filter. when labeled probes are hybridized to these immobilized rna molecules, their presence can be visualized by autoradiography. here we describe northern hybridization using radioactively labeled riboprobes to show circadian expression ...200717417020
isolation of total rna from neurospora mycelium.in filamentous fungi, including the model organism neurospora crassa, plentiful biological tissue from which rna can be extracted may be obtained by allowing fungal spores to germinate and form a mycelium in liquid culture. the mycelium constitutes a mosaic of multinuclear, tubular filaments known as hyphae or mycelia. in general, when exposed to air, fungal hyphae quickly start to develop spores, which are often colorful. however, when submerged in liquid under rapid agitation large amounts of ...200717417016
novel strategies for identification of clock genes in neurospora with insertional mutagenesis.as the molecular mechanism of circadian clocks has reached high complexity, the fungal model system, neurospora crassa, is increasingly important for clock research. it offers the possibility of extensive biochemical experimentation and thorough description of circadian properties. realization of the full potential is dependent on efficient, high-throughput methods. we have combined several protocols to develop abundant and inexpensive production of mutants, and subsequent identification of the ...200717417009
rhythmic conidiation in neurospora crassa.in the filamentous fungus neurospora crassa the production of asexual spores (conidia) is regulated by its circadian clock. when the fungus is grown on a thin layer of agar medium in long growth tubes (so-called "race tubes"), restricting its growth to one direction only, bright orange bands are clearly visible. this banding pattern persists with a periodicity of approx 24 h in the absence of any environmental stimuli. the bands are formed by alternating zones of nonsporulating mycelium and myce ...200717417000
quelling: post-transcriptional gene silencing guided by small rnas in neurospora crassa.the filamentous fungus neurospora crassa is a model organism for the study of gene silencing. the most characterized gene silencing mechanism in this ascomycete is quelling, which occurs at the post-transcriptional level. quelling is triggered by the introduction of transgenes and results in silencing of both transgenes and cognate endogenous mrnas. quelling is related to co-suppression, observed in plants, and rna interference in animals; it requires an argonaute protein and acts by generating ...200717395524
the response regulator rrg-1 functions upstream of a mitogen-activated protein kinase pathway impacting asexual development, female fertility, osmotic stress, and fungicide resistance in neurospora crassa.two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, archaea, fungi, slime molds, and plants. here, we characterize rrg-1, a response regulator protein from the filamentous fungus neurospora crassa. the cell lysis phenotype of delta rrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protei ...200717392518
the external alternative nad(p)h dehydrogenase nde3 is localized both in the mitochondria and in the cytoplasm of neurospora crassa.the filamentous fungus neurospora crassa has a branched respiratory chain. several alternative dehydrogenases, aside from the canonical complex i enzyme, are involved in the oxidation of nad(p)h substrates. based on homology searches in the fungal genome, we have tentatively identified one of these proteins. the corresponding gene was inactivated by the generation of repeat-induced point mutations and a null-mutant strain was isolated. this mutant is deficient in the oxidation of cytosolic nadh, ...200717379240
pentaketide resorcylic acid synthesis by type iii polyketide synthase from neurospora crassa.type iii polyketide synthases (pkss) are responsible for aromatic polyketide synthesis in plants and bacteria. genome analysis of filamentous fungi has predicted the presence of fungal type iii pkss, although none have thus far been functionally characterized. in the genome of neurospora crassa, a single open reading frame, ncu04801.1, annotated as a type iii pks was found. in this report, we demonstrate that ncu04801.1 is a novel type iii pks catalyzing the synthesis of pentaketide alkylresorcy ...200717374612
a double-stranded-rna response program important for rna interference efficiency.when recognized by the rna interference (rnai) pathway, double-stranded rna (dsrna) produced in eukaryotic cells results in posttranscriptional gene silencing. in addition, dsrna can trigger the interferon response as part of the immune response in vertebrates. in this study, we show that dsrna, but not short interfering rna (sirna), induces the expression of qde-2 (an argonaute gene) and dcl-2 (a dicer gene), two central components of the rnai pathway in the filamentous fungus neurospora crassa ...200717371837
complexity of the neurospora crassa circadian clock system: multiple loops and oscillators.organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. we review evidence from neurospora crassa that suggests that the circadian clock is organized as a network of genes and proteins that form coupled evening- and morning-specific oscillatory loops that can function autonomously, respond differently to environmental inputs, and regulate phase-specific outputs. there is also evidence for coupled morning and evening oscillator ...200718419292
circadian entrainment of neurospora crassa.the circadian clock evolved under entraining conditions, yet most circadian experiments and much circadian theory are built around free-running rhythms. the interpretation of entrainment experiments is certainly more complex than that of free-running rhythms due to the relationship between exogenous and endogenous cycles. here, we systematically describe entrainment in the simplest of the traditional eukaryotic model systems in circadian research, neurospora crassa. this fungus forms a mass of s ...200718419284
two zinc-cluster transcription factors control induction of alternative oxidase in neurospora crassa.the alternative oxidase transfers electrons from ubiquinol to molecular oxygen, providing a mechanism for bypassing the later steps of the standard cytochrome-mediated electron transport chain. the enzyme is found in an array of organisms and in many cases is known to be produced in response to perturbations of the standard chain. alternative oxidase is encoded in the nucleus but functions in the inner mitochondrial membrane. this implies the existence of a retrograde regulation pathway for comm ...200718073419
continuous enzymatic assay for histone lysine methyltransferases.we describe a continuous peptide methylation assay using the neurospora crassa dim-5 histone h3 lysine 9 (h3k9) methyltransferase as a model system. the assay uses streptavidin flashplates coated with target peptide. since no washing and pipeting steps were required after the addition of the enzyme/s-adenosyl-l-methionine (adomet) mixture to the microplate, a continuous readout of the reaction progress was possible. we show that this assay is highly reproducible (with errors in the order of +/- ...200718072589
circadian rhythm in the pink-orange bread mould neurospora crassa: for what? 200717954967
quantitative trait loci for the circadian clock in neurospora crassa.neurospora crassa has been a model organism for the study of circadian clocks for the past four decades. among natural accessions of neurospora crassa, there is significant variation in clock phenotypes. in an attempt to investigate natural allelic variants contributing to quantitative variation, we used a quantitative trait loci mapping approach to analyze three independent mapping populations whose progenitors were collected from geographically isolated locations. two circadian clock phenotype ...200717947430
systems biology of the neurospora biological clock.a major challenge of systems biology is explaining complex traits, such as the biological clock, in terms of the kinetics of macromolecules. the clock poses at least four challenges for systems biology: (i) identifying the genetic network to explain the clock mechanism quantitatively; (ii) specifying the clock's functional connection to a thousand or more genes and their products in the genome; (iii) explaining the clock's response to light and other environmental cues; and (iv) explaining how t ...200717907673
the transcription of the gene for iso-orotate decarboxylase (idcase), an enzyme of the thymidine salvage pathway, is downregulated in the pregc mutant strain of neurospora crassa grown under phosphate starvation.the preg gene encodes a cyclin-like protein that is implicated in the derepression of nucleases and phosphatases that scavenge phosphate from the environment. to better understand the regulatory role of the preg gene product, the differential display reverse transcriptase - polymerase chain reaction was used to isolate transcripts differentially expressed in the pregc mutant strain of the mold neurospora crassa grown under phosphate starvation, at ph 7.8. two transcripts, whose differential expr ...200717898858
ionic currents and ion fluxes in neurospora crassa hyphae.voltage dependence of ionic currents and ion fluxes in a walled, turgor-regulating cell were measured in neurospora crassa. the hyphal morphology of the model organism neurospora simplifies cable analysis of ionic currents to determine current density for quantitative comparisons with ion fluxes. the ion fluxes were measured directly and non-invasively with self-referencing ion-selective microelectrodes. four ions (h(+), ca(2+), k(+), and cl(-)) were examined. h(+) net uptake and ca(2+) net rele ...200717898420
supramolecular organization of the respiratory chain in neurospora crassa mitochondria.the existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. however, its functional significance is still poorly understood. the availability of many deletion mutants in complex i (nadh:ubiquinone oxidoreductase) of neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. herein, we describe the role of complex i in assembly of respiratory complexes and ...200717873079
migration cues induce chromatin alterations.directed cell migration is a property central to multiple basic biological processes. here, we show that directed cell migration is associated with global changes in the chromatin fiber. polarized posttranslational changes in histone h1 along with a transient decrease in h1 mobility were detected in cells facing the scratch in a wound healing assay. in parallel to the changes in h1, the levels of the heterochromatin marker histone h3 lysine 9 tri-methylation were elevated. interestingly, reducti ...200717822403
in silico mining in expressed sequences of neurospora crassa for identification and abundance of microsatellites.in the present study, 3217 unigene sequences of neurospora crassa downloaded from the national center for biotechnology information (ncbi) were mined for the identification of microsatellites or simple sequence repeats (ssrs). a total of 287 ssrs detected gives density of 1ssr/14.6 kb of 4187.86 kb sequences mined suggests that only 250 (7.8%) of sequences contained ssrs. depending on the repeat units, the length of ssrs ranged from 14 to 17 bp for mono-, 14 to 48 bp for di-, 18 to 90 bp for tri ...200716875812
so, a protein involved in hyphal fusion in neurospora crassa, localizes to septal plugs.the colony of a filamentous ascomycete fungus typically grows as a multinucleate syncytium. while this syncytial organization has developmental advantages, it bears the risk of extensive damage caused by local injury of hyphae. loss of cytoplasm in injured hyphae is restricted by the fast and efficient sealing of the central pores of hyphal crosswalls, or septa, by a peroxisome-derived organelle called the woronin body. the formation of septal plugs is also associated with development and leads ...200717099082
multiple layers of temporal and spatial control regulate accumulation of the fruiting body-specific protein app in sordaria macrospora and neurospora crassa.during fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. we have isolated a protein from the ascomycete sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. the protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant perithecial protein). app transcript occurs only after the onset of sexual development; however, the formation of ascospores is not a prerequisite for app ...200717092746
genetic basis of the ovc phenotype of neurospora: identification and analysis of a 77 kb deletion.the ovc mutant of neurospora crassa accumulates more carotenoids than the wild type in the light, is sensitive to high osmotic pressure and exhibits an altered aerial development. the three traits are complemented by a single gene, cut-1, but only the two latter are exhibited by a mutant of this gene carrying a premature stop mutation. targeted cut-1 deletion results in a normal carotenoid content, confirming the involvement of at least a second gene in the carotenoid-overproducing phenotype of ...200717082948
rapid genetic mapping in neurospora crassa.forward genetic analysis is the most broadly applicable approach to discern gene functions. however, for some organisms like the filamentous ascomycete neurospora crassa, genetic mapping frequently represents a limiting step in forward genetic approaches. we describe an efficient method for genetic mapping in n. crassa that makes use of a modified bulked segregant analysis and pcr-based molecular markers. this method enables mapping with progeny from a single cross and requires only 90 pcr ampli ...200717056287
mutation and divergence of the phospholipase c gene in neurospora crassa.in the fungus neurospora crassa we have used rip to obtain a presumptive null mutation of the phospholipase c-1 gene, thought to be important in intracellular calcium signaling, notably maintenance of the tip-high calcium gradient. the mutant is viable but has slow, aberrant growth and branching. hence plc-1 is not required for polar growth at the tip, but is necessary to modulate growth to give normal form. the mutant has residual plc activity suggesting that this enzyme function can be provide ...200717157541
ontogeny of the spitzenkörper in germlings of neurospora crassa.the spitzenkörper (spk) is a highly dynamic and pleomorphic complex located at the hyphal apex of filamentous fungi. most studies revealing the structure and behavior of the spk have been conducted on mature vegetative hyphae of filamentous fungi, including both main leading hyphae and branches. however, these reports do not address whether the observations can be extended to germ tubes. by enhanced phase-contrast video-microscopy and laser scanning confocal microscopy we have analyzed the intra ...200717127085
os-2 mitogen activated protein kinase regulates the clock-controlled gene ccg-1 in neurospora crassa.os-2 map kinase is involved in osmoadaptation in neurospora crassa. clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an os-2 dependent manner. in contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. these results suggest that os-2 participates in the regulation of certain circadian-clock output genes.200717986782
circadian rhythmicity mediated by temporal regulation of the activity of p38 mapk.circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. little is known about the output pathways. in this study, we show that the neurospora crassa osmosensing mapk pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. rhythmic activation of the h ...200717984065
metabolism of androst-4-en-3,17-dione by the filamentous fungus neurospora crassa.microbial transformation of androst-4-en-3,17-dione (ad; i) using neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (ii), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (iii), 7alpha-hydroxyandrost-4-en-3,17-dione (iv), 9alpha-hydroxyandrost-4-en-3,17-dione (v), 14alpha-hydroxyandrost-4-en-3,17-dione (vi), and androst-4,6-dien-3,17-dione (vii). the steroid products were assigned by interpretation of their spectral data such as (1)h nmr, (13)c nmr, ftir, an ...200817981313
simulating dark expressions and interactions of frq and wc-1 in the neurospora circadian clock.circadian rhythms are considered to play an essential part in the adaptation of organisms to their environments. the occurrence of circadian oscillations appears to be based on the presence of transcriptional-translational negative feedback loops. in neurospora crassa, the protein frequency (frq) is part of such a negative feedback loop apparently by a direct interaction with its transcription factor white collar-1 (wc-1). based on the observation that nuclear frq levels are significantly lower ...200817965132
fully codon-optimized luciferase uncovers novel temperature characteristics of the neurospora clock.we report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in neurospora crassa. this reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, show ...200817766461
microtubule dynamics and the role of molecular motors in neurospora crassa.live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of neurospora crassa expressing beta-tubulin-gfp. microtubule polymerization rates in hyphae of n. crassa were much faster than those previously reported in any other eukaryotic organism. in order to address the roles of motor proteins in microtubule dynamic instability in n. crassa, the microtubule-motor mutant strains, deltankin and ro-1, were examined. polymerization and de ...200818069024
the rna-dependent rna polymerase essential for post-transcriptional gene silencing in neurospora crassa interacts with replication protein a.post-transcriptional gene silencing (ptgs) pathways play a role in genome defence and have been extensively studied, yet how repetitive elements in the genome are identified is still unclear. it has been suggested that they may produce aberrant transcripts (arna) that are converted by an rna-dependent rna polymerase (rdrp) into double-stranded rna (dsrna), the essential intermediate of ptgs. however, how rdrp enzymes recognize aberrant transcripts remains a key question. here we show that in neu ...200818048414
titration of repeat-induced point mutation (rip) by chromosome segment duplications in neurospora crassa.repeat-induced point mutation (rip) is a hypermutational process that alters duplicated dna sequences in neurospora crassa. in previous studies, five of six large ( > 100 kb) chromosome segment duplications (dp's) examined were shown to dominantly suppress rip in smaller (< 5 kb) duplications. the suppressor duplications were > 270 kb, whereas the lone non-suppressor duplication was approximately 117 kb. we have now screened another 33 duplications and found 29 more suppressors and four more non ...200818046508
the evolution of the pheromonal signal system and its potential role for reproductive isolation in heterothallic neurospora.comparative sequencing studies among a wide range of taxonomic groups, including fungi, provide the overall pattern that reproductive genes evolve more rapidly than other genes, and this divergence is believed to be important in the establishment of reproductive barriers between species. in this study, we investigated the molecular evolution of the pheromone receptor genes pre-1 and pre-2 of strains belonging to 12 and 13 heterothallic taxa, respectively, of the model genus neurospora. furthermo ...200818024989
genetic analysis of chk1 and chk2 homologues revealed a unique cross talk between atm and atr pathways in neurospora crassa.dna damage checkpoint is an important mechanism for organisms to maintain genome integrity. in neurospora crassa, mus-9 and mus-21 are homologues of atr and atm, respectively, which are pivotal factors of dna damage checkpoint in mammals. a n. crassa clock gene prd-4 has been identified as a chk2 homologue, but its role in dna damage response had not been elucidated. in this study, we identified another chk2 homologue and one chk1 homologue from the n. crassa genome database. as disruption of th ...200818790091
systems biology of the clock in neurospora crassa.a model-driven discovery process, computing life, is used to identify an ensemble of genetic networks that describe the biological clock. a clock mechanism involving the genes white-collar-1 and white-collar-2 (wc-1 and wc-2) that encode a transcriptional activator (as well as a blue-light receptor) and an oscillator frequency (frq) that encodes a cyclin that deactivates the activator is used to guide this discovery process through three cycles of microarray experiments. central to this discover ...200818769678
toward predicting self-splicing and protein-facilitated splicing of group i introns.in the current era of massive discoveries of noncoding rnas within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. although studies of individual group i introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group i introns sequenced to date. here, the self-splicing activities of 12 group i introns from various organisms ...200818768647
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