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enzymes with extra talents: moonlighting functions and catalytic promiscuity.recent studies of moonlighting functions and catalytic promiscuity provide insights into the structural and mechanistic bases of these phenomena. moonlighting proteins that are highlighted include gephyrin, the neurospora crassa tyrosyl trna synthetase, phosphoglucose isomerase, and cytochrome c. new insights into catalytic promiscuity are provided by studies of aminoglycoside kinase (3') type iiia, tetrachlorohydroquinone dehalogenase, and aldolase antibody 38c2.200312714060
the methylated component of the neurospora crassa genome.cytosine methylation is common, but not ubiquitous, in eukaryotes. mammals and the fungus neurospora crassa have about 2-3% of cytosines methylated. in mammals, methylation is almost exclusively in the under-represented cpg dinucleotides, and most cpgs are methylated whereas in neurospora, methylation is not preferentially in cpg dinucleotides and the bulk of the genome is unmethylated. dna methylation is essential in mammals but is dispensable in neurospora, making this simple eukaryote a favou ...200312712205
histone h1 is required for proper regulation of pyruvate decarboxylase gene expression in neurospora crassa.we show that neurospora crassa has a single histone h1 gene, hh1, which encodes a typical linker histone with highly basic n- and c-terminal tails and a central globular domain. a green fluorescent protein-tagged histone h1 chimeric protein was localized exclusively to nuclei. mutation of hh1 by repeat-induced point mutation (rip) did not result in detectable defects in morphology, dna methylation, mutagen sensitivity, dna repair, fertility, rip, chromosome pairing, or chromosome segregation. ne ...200312684383
trimethylated lysine 9 of histone h3 is a mark for dna methylation in neurospora crassa.besides serving to package nuclear dna, histones carry information in the form of a diverse array of post-translational modifications. methylation of histones h3 and h4 has been implicated in long-term epigenetic 'memory'. dimethylation or trimethylation of lys4 of histone h3 (h3 lys4) has been found in expressible euchromatin of yeasts and mammals. in contrast, methylation of lys9 of histone h3 (h3 lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin o ...200312679815
what's in the genome of a filamentous fungus? analysis of the neurospora genome sequence.the german neurospora genome project has assembled sequences from ordered cosmid and bac clones of linkage groups ii and v of the genome of neurospora crassa in 13 and 12 contigs, respectively. including additional sequences located on other linkage groups a total of 12 mb were subjected to a manual gene extraction and annotation process. the genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. the analysis of the 3218 iden ...200312655011
a hex-1 crystal lattice required for woronin body function in neurospora crassa.the woronin body is a dense-core vesicle specific to filamentous ascomycetes (euascomycetes), where it functions to seal the septal pore in response to cellular damage. the hex-1 protein self-assembles to form this solid core of the vesicle. here, we solve the crystal structure of hex-1 at 1.8 a, which provides the structural basis of its self-assembly. the structure reveals the existence of three intermolecular interfaces that promote the formation of a three-dimensional protein lattice. consis ...200312640443
synthesis of signals for de novo dna methylation in neurospora crassa.most 5-methylcytosine in neurospora crassa occurs in a:t-rich sequences high in tpa dinucleotides, hallmarks of repeat-induced point mutation. to investigate how such sequences induce methylation, we developed a sensitive in vivo system. tests of various 25- to 100-bp synthetic dna sequences revealed that both t and a residues were required on a given strand to induce appreciable methylation. segments composed of (taaa)(n) or (ttaa)(n) were the most potent signals; 25-mers induced robust methyla ...200312640122
on the independence of barrage formation and heterokaryon incompatibility in neurospora crassa.a barrage is a line or zone of demarcation that may develop at the interface where genetically different fungi meet. barrage formation represents a type of nonself recognition that has often been attributed to the heterokaryon incompatibility system, which limits the co-occurrence of genetically different nuclei in the same cytoplasm during the asexual phase of the life cycle. while the genetic basis of the heterokaryon incompatibility system is well characterized in neurospora crassa, barrage f ...200312620257
role for antisense rna in regulating circadian clock function in neurospora crassa.the prevalence of antisense rna in eukaryotes is not known and only a few naturally occurring antisense transcripts have been assigned a function. however, the recent identification of a large number of putative antisense transcripts strengthens the view that antisense rnas might affect a wider variety of processes than previously thought. here we show that in the model organism neurospora crassa entrainment of the circadian clock, which is critical for the correct temporal expression of genes a ...200312607002
the qde-3 homologue recq-2 co-operates with qde-3 in dna repair in neurospora crassa.the post-transcriptional gene silencing mechanism in neurospora crassa, called quelling, was shown to involve the products of three genes termed quelling-defective. a homologue to the qde-3 gene encoding a putative recq-type dna helicase was isolated and was named recq-2. characterisation of the recq-2 gene has revealed that it is not involved in quelling, but may co-operate with the qde-3 gene product in a pathway that repairs damage to dna caused by the chemical mutagens methyl methanesulfonat ...200312589473
roles for white collar-1 in circadian and general photoperception in neurospora crassa.the transcription factors white collar-1 (wc-1) and white collar-2 (wc-2) interact to form a heterodimeric complex (wcc) that is essential for most of the light-mediated processes in neurospora crassa. wcc also plays a distinct non-light-related role as the transcriptional activator in the frequency (frq)/wcc feedback loop that is central to the n. crassa circadian system. although an activator role was expected for wc-1, unanticipated phenotypes resulting from some wc-1 alleles prompted a close ...200312586700
the internal alternative nadh dehydrogenase of neurospora crassa mitochondria.an open reading frame homologous with genes of non-proton-pumping nadh dehydrogenases was identified in the genome of neurospora crassa. the 57 kda nadh:ubiquinone oxidoreductase acts as internal (alternative) respiratory nadh dehydrogenase (ndi1) in the fungal mitochondria. the precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one fad molecule as a prosthetic group. it catalyses specifically the oxidation of nadh. western blot analysis of fungal mi ...200312556227
the oligomeric state, complex formation, and chaperoning activity of hsp70 and hsp80 of neurospora crassa.molecular chaperones perform vital cellular functions under normal growth conditions and protect cells against stress-induced damage. the stress proteins hsp70 and hsp80 of neurospora crassa were extracted from heat-shocked mycelium, purified to near homogeneity, and examined with respect to their oligomeric state, complex formation, and chaperoning properties. their oligomeric state was assessed by dynamic light-scattering measurements, and both hsp70 and hsp80 were observed to form a range of ...200212555813
detection of unpaired dna at meiosis results in rna-mediated silencing.during meiosis, homologous chromosomes must pair in order to permit recombination and correct chromosome segregation to occur. two recent papers show that meiotic pairing is also important for correct gene expression during meiosis. they describe data for the filamentous fungus neurospora crassa that show that a lack of pairing generated by ectopic integration of genes can result in silencing of genes expressed during meiosis. this can result in aberrant meioses whose defects are specific to the ...200312539234
expression of protein phosphatase 1 during the asexual development of neurospora crassa.we cloned and sequenced the cdna and the gene encoding the catalytic subunit of protein phosphatase 1 from the filamentous fungus neurospora crassa. the gene, designated ppp-1 (phosphoprotein phosphatase 1), was mapped by restriction fragment length polymorphism to linkage group iii, in the vicinity of con-7 and trp-1. the expression of the gene was monitored by reverse transcriptase and polymerase chain reactions, by western blotting, and by protein phosphatase activity assays in synchronized c ...200312524044
detection of mutagenic activities of various pesticides in neurospora crassa. 200312478420
an ensemble method for identifying regulatory circuits with special reference to the qa gene cluster of neurospora crassa.a chemical reaction network for the regulation of the quinic acid (qa) gene cluster of neurospora crassa is proposed. an efficient monte carlo method for walking through the parameter space of possible chemical reaction networks is developed to identify an ensemble of deterministic kinetics models with rate constants consistent with rna and protein profiling data. this method was successful in identifying a model ensemble fitting available rna profiling data on the qa gene cluster.200212477937
multiple functions of mfa-1, a putative pheromone precursor gene of neurospora crassa.a putative pheromone precursor gene of neurospora crassa, mfa-1 (which encodes mating factor a-1), was identified as the most abundant clone in starved mycelial and perithecial cdna libraries. northern analysis demonstrated high mfa-1 expression in all mating type a tissues and suggested low expression levels in mat a tissues. the mfa-1 gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3' untranslated region (3' utr). the predic ...200212477799
shared and independent roles for a galpha(i) protein and adenylyl cyclase in regulating development and stress responses in neurospora crassa.growth and development are regulated using cyclic amp (camp)-dependent and -independent pathways in neurospora crassa. the cr-1 adenylyl cyclase mutant lacks detectable camp and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. evidence suggests that the heterotrimeric galpha protein gna-1 is a direct positive regulator of adenylyl cyclase. deltagna-1 str ...200212456011
neurospora clock-controlled gene 9 (ccg-9) encodes trehalose synthase: circadian regulation of stress responses and development.the circadian clock of neurospora crassa regulates the rhythmic expression of a number of genes encoding diverse functions which, as an ensemble, are adaptive to life in a rhythmic environment of alternating levels of light and dark, warmth and coolness, and dryness and humidity. previous differential screens have identified a number of such genes based solely on their cycling expression, including clock-controlled gene 9 (ccg-9). sequence analysis now shows the predicted ccg-9 polypeptide to be ...200212455969
distinct signaling pathways from the circadian clock participate in regulation of rhythmic conidiospore development in neurospora crassa.several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in neurospora crassa. however, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in n. crassa promotes daily rhythms in expression of known developmental genes and of conidiation. we anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and ...200212455961
morphological alterations induced by cold-shock in neurospora crassa.the effects of cold-shock on neurospora crassa was here investigated. in cold-shock treated hyphae (6 h at 4 degrees c) the branching pattern was changed and dichotomy as well as a fan-like-pattern structure were temporarily detected at the tips. when exogenous ca(2+) (500 mm) was added to the hyphae, these morphological alterations were not detected. in addition, the protein electrophoretic profile of cultures submitted to cold-shock was different from that of cultures kept at 30 degrees c. aft ...200212442300
a non-modular type b feruloyl esterase from neurospora crassa exhibits concentration-dependent substrate inhibition.feruloyl esterases, a subclass of the carboxylic acid esterases (ec 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. the enzymes have been classified as type a or type b, based on their substrate specificity for aromatic moieties. we show that neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates ...200312435269
an ip3-activated ca2+ channel regulates fungal tip growth.hyphal extension in fungi requires a tip-high ca(2+) gradient, which is generated and maintained internally by inositol (1,4,5)-trisphosphate (ip(3))-induced ca(2+) release from tip-localized vesicles and subapical ca(2+) sequestration. using the planar bilayer method we demonstrated the presence of two types of ip(3)-activated ca(2+) channels in neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). on sucrose density gradients the lo ...200212432087
the effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of neurospora crassa.we have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. based on video-enhanced dic light microscopy, the mature, growing hyphae of n. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (i) and three subapical regions (ii, iii, and iv). a well-defined spitzenkörper d ...200212409101
a fourier transform infrared study of neurospora rhodopsin: similarities with archaeal rhodopsins.the nop-1 gene from the eukaryote neurospora crassa, a filamentous fungus, has recently been shown to encode an archaeal rhodopsin-like protein nop-1. to explore the functional mechanism of nop-1 and its possible similarities to archaeal and visual rhodopsins, static and time-resolved fourier transform infrared difference spectra were measured from wild-type nop-1 and from a mutant containing an asp-->glu substitution in the schiff base (sb) counterion, asp131 (d131e). several conclusions could ...200212403457
characterization of neurospora crassa tom40-deficient mutants and effect of specific mutations on tom40 assembly.the tom complex (translocase of the outer mitochondrial membrane) is responsible for the recognition of mitochondrial preproteins synthesized in the cytosol and for their translocation across or into the outer mitochondrial membrane. tom40 is the major component of the tom complex and forms the translocation pore. we have created a tom40 mutant of neurospora crassa and have demonstrated that the gene is essential for the viability of the organism. mitochondria with reduced levels of tom40 were d ...200312399467
a new wc-1 mutant of neurospora crassa shows unique light sensitivity in the circadian conidiation rhythm.a new clock mutant ( rhy-2) was isolated by dna insertion mutagenesis using a plasmid that contains a region located upstream of the cmd gene in the genome of neurospora crassa. this mutant is arrhythmic with regard to conidiation in continuous darkness but rhythmic under a light-dark cycle. after plasmid rescue from genomic dna of the rhy-2 strain, the insertion was localized to the gene white collar-1 ( wc-1). plasmid dna was inserted 3' to the sequence encoding the polyglutamine region of the ...200212242499
nonself recognition is mediated by het-c heterocomplex formation during vegetative incompatibility.nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. in neurospora crassa, het-c is one of 11 het loci. three allelic specificity groups, termed het-c(or), het-c(pa) and het-c(gr), exist in natural populations. heterokaryons or partial diploids that contain het-c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (hcd). using epitope-tagged het-c, we ...200212234924
live-cell imaging of vegetative hyphal fusion in neurospora crassa.the process of hyphal fusion (anastomosis) in growing colonies of neurospora crassa, stained with the membrane-selective dyes fm1-43 and fm4-64, was visualized by confocal microscopy. time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facili ...200212223195
from nadh to ubiquinone in neurospora mitochondria.the respiratory chain of the mitochondrial inner membrane includes a proton-pumping enzyme, complex i, which catalyses electron transfer from nadh to ubiquinone. this electron pathway occurs through a series of protein-bound prosthetic groups, fmn and around eight iron-sulfur clusters. the high number of polypeptide subunits of mitochondrial complex i, around 40, have a dual genetic origin. neurospora crassa has been a useful genetic model to characterise complex i. the characterisation of mutan ...200212206913
the art and design of genetic screens: filamentous fungi.in the 1940s, screens for metabolic mutants of the filamentous fungus neurospora crassa established the fundamental, one-to-one relationship between a gene and a specific protein, and also established fungi as important genetic organisms. today, a wide range of filamentous species, which represents a billion years of evolutionary divergence, is used for experimental studies. the developmental complexity of these fungi sets them apart from unicellular yeasts, and allows the development of new scr ...200212209143
blue light modulation of ion transport in the slime mutant of neurospora crassa.blue light is the primary entrainment signal for a number of developmental and morphological processes in the lower eucaryote neurospora crassa. blue light regulates photoactivation of carotenoid synthesis, conidiation, phototropism of perithecia and circadian rhythms. changes in the electrical properties of the plasma membrane are one of the fastest responses to blue light irradiation. to enable patch-clamp studies on light-induced ion channel activity, the wall-less slime mutant was used. patc ...200212181612
overexpression of white collar-1 (wc-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in neurospora crassa.many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. the white collar-1 (wc-1) protein is required for all known blue-light responses in neurospora crassa. in response to light, wc-1 levels increase, and the protein is transiently phosphorylated. to test the hypothesis that the increase in wc-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatme ...200212180913
the neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock.pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. to begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species neurospora crassa. one gene, expressed exclusively in mat a strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by kex2 processing sites. strains of the opposite mating type, mat a, express a pheromone precursor g ...200212139624
neurospora crassa supersuppressor mutants are amber codon-specific.neurospora crassa has 10 mapped supersuppressor (ssu) genes. in vivo studies indicate that they suppress amber (uag) premature termination mutations but the spectrum of their functions remains to be elucidated. we examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. we tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature uaa, uga, or uag terminators. all mutants except ssu-3 suppressed uag codo ...200212135572
light reception and circadian behavior in 'blind' and 'clock-less' mutants of neurospora crassa.the filamentous fungus neurospora crassa is a model organism for the genetic dissection of blue light photoreception and circadian rhythms. white collar-1 (wc-1) and wc-2 are considered necessary for all light responses, while frequency (frq) is required for light-regulated asexual development (conidia formation); without any of the three, self-sustained (circadian) rhythmicity in constant conditions fails. here we show that light-regulated and self-sustained development occur in the individual ...200212110577
domain movements of plasma membrane h(+)-atpase: 3d structures of two states by electron cryo-microscopy.h(+)-atpase is a p-type atpase that transports protons across membranes using the energy from atp hydrolysis. this hydrolysis is coupled to a conformational change between states of the protein, in which the proton-binding site is alternately accessible to the two sides of the membrane with an altered affinity. when isolated from neurospora crassa, h(+)-atpase is a 600 kda hexamer of identical 100 kda polypeptides. we have obtained the three-dimensional structures of both ligand-free and mg(2+)/ ...200212110571
white collar-1, a circadian blue light photoreceptor, binding to the frequency promoter.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 co ...200212098706
white collar-1, a dna binding transcription factor and a light sensor.blue light regulates many physiological processes in fungi, but their photoreceptors are not known. in neurospora crassa, all light responses depend on the per-arnt-sim (pas) domain-containing transcription factor white collar-1 (wc-1). by removing the wc-1 light, oxygen, or voltage domain, a specialized pas domain that binds flavin mononucleotide in plant phototropins, we show that light responses are abolished, including light entrainment of the circadian clock. however, the wc-1-mediated dark ...200212098705
a dead-box protein functions as an atp-dependent rna chaperone in group i intron splicing.the neurospora crassa cyt-18 protein, the mitochondrial tyrosyl-trna synthetase, functions in splicing group i introns by inducing formation of the catalytically active rna structure. here, we identified a dead-box protein (cyt-19) that functions in concert with cyt-18 to promote group i intron splicing in vivo and vitro. cyt-19 does not bind specifically to group i intron rnas and instead functions as an atp-dependent rna chaperone to destabilize nonnative rna structures that constitute kinetic ...200212086675
mitochondrial dynamics in filamentous fungi.mitochondria are essential organelles of eukaryotic cells. they grow continuously throughout the cell cycle and are inherited by daughter cells upon cell division. inheritance of mitochondria and maintenance of mitochondrial distribution and morphology require active transport of the organelles along the cytoskeleton and depend on membrane fission and fusion events. many of the molecular components and cellular mechanisms mediating these complex processes have been conserved during evolution acr ...200212081462
protein import into mitochondria of neurospora crassa.biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mitochondrial structure and function. import and sorting of these preproteins is a multistep process facilitated by complex proteinaceous machineries located in the mitochondrial outer and inner membranes. the translocase of the mitochondrial outer membrane, the tom complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel f ...200212081461
nht2, a copia ltr retrotransposon from a conditionally dispensable chromosome in nectria haematococca.in the plant pathogenic ascomycete nectria haematococca mating population (mp) vi, the conditionally dispensable chromosomes are unstable during sexual reproduction. during mapping of such a chromosome, three dispersed repeats were identified. nht2, one of these repeated elements, is a long terminal repeat (ltr) retrotransposon that is 5.9 kb in length. its deduced amino acid sequence is homologous to the four enzymatic domains characteristic of copia retrotransposons, but it contains multiple s ...200212073091
multilocus self-recognition systems in fungi as a cause of trans-species polymorphism.trans-species polymorphism, meaning the presence of alleles in different species that are more similar to each other than they are to alleles in the same species, has been found at loci associated with vegetative incompatibility in filamentous fungi. if individuals differ at one or more of these loci (termed het for heterokaryon), they cannot form stable heterokaryons after vegetative fusion. at the het-c locus in neurospora crassa and related species there is clear evidence of trans-species pol ...200212072460
circadian period lengths of lipid synthesis mutants (cel, chol-1) of neurospora show defective temperature, but intact ph-compensation.the influence of extracellular ph on the circadian sporulation rhythm of neurospora crassa has been investigated for the mutants chol-1 and cel. both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. the chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. we investigated the possible correlation between loss of temperature- and ph-compensation in cel and c ...200212069035
[superoxide dismutase and catalase activities in carotenoid-synthesizing fungi blakeslea trispora and neurospora crassa under the oxidative stress].the addition of menadione into the medium during cultivation of neurospora crassa in the dark activated its constitutive superoxide dismutase. exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. superoxide dismutase activity in the mixed (+/-) mycelium of blakeslea trispora synthesizing beta-carotene in the dark was much lower than that in neurospora crassa. the superoxide dismutase activity further decreased in oxidative s ...201312068573
the many faces of histone lysine methylation.diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity. within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone h3 am ...200212067650
neurospora crassa catalases, singlet oxygen and cell differentiation.the morphogenetic transitions of the n. crassa asexual life cycle are responses to a hyperoxidant state in which probably singlet oxygen is generated. induction of catalase activity and catalase oxidation by singlet oxygen are consequences of this recurrent hyperoxidant state. here the biochemical properties and regulation of two large monofunctional catalases are reviewed, and a new catalase-peroxidase gene and activity is described. catalase-3 is associated to growing and catalase-1 to non-gro ...200612033445
minimal promoter for the nad+-specific glutamate dehydrogenase gene of neurospora crassa.the expression of the nad+-specific glutamate dehydrogenase (nad-gdh) gene of neurospora crassa is subject to catabolite repression. to identify the minimal sequence necessary for promoter function, the 5'-flanking region of the nad-gdh gene was screened for potential protein-binding sites. fragments of dna, containing sequences upstream from the atg initiation codon, were employed as probes of southwestern blots of total cellular protein from cells grown in media promoting repression and induct ...200211989713
3-carboxy-cis,cis-muconate lactonizing enzyme from neurospora crassa: mad phasing with 80 selenomethionines.the structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from neurospora crassa was determined at 3.0 a resolution. phase information was derived from a multiwavelength anomalous dispersion (mad) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. however, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. eventually, 80 selenium sites ...200211976482
isolation and characterization of tightly coupled mitochondria from wild type and nap mutant neurospora crassa.a fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap-mutant neurospora crassa cells harvested at different growth stages. the isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal adp/o ratios upon oxidation of krebs cycle intermediates and exogenous nadh. they contained the fully competent respirator ...200211952424
involvement of small rnas and role of the qde genes in the gene silencing pathway in neurospora.small rna molecules have been found to be specifically associated with posttranscriptional gene silencing (ptgs) in both plants and animals. here, we find that small sense and antisense rnas are also involved in ptgs in neurospora crassa. the accumulation of these rna molecules depends on the presence of functional qde-1 and qde-3 genes previously shown to be essential for gene silencing, but does not depend on a functional qde-2, indicating that this gene is involved in a downstream step of the ...200211937487
the structure of neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase.muconate lactonizing enzymes (mles) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. there are three different mle classes unrelated by evolution. we present the x-ray structure of a eukaryotic mle, neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (nccmle) at 2.5 a resolution, with a seven-bladed beta propeller fold. it is related neither to bacterial mles nor t ...200211937053
quelling in neurospora crassa. 200211931228
transformation in heterokaryons of neurospora crassa is nuclear rather than cellular phenomenon.in neurospora crassa, multinucleate macroconidia are used for genetic transformation. the barrier for such a transformation can be either at the cell membrane level or at the nuclear membrane level. for assessment of these possibilities, a forced heterokaryon (containing two genetically marked nuclei and auxotrophic for histidine) of neurospora crassa was transformed with a plasmid containing his-3+ gene. the transformants, which could grow without histidine supplementation, were then resolved i ...200211927980
evidence for dominant suppression of repeat-induced point mutation (rip) in crosses with the wild-isolated neurospora crassa strains sugartown and adiopodoume-7.a convenient assay to score repeat-induced point mutation (rip) in neurospora employs the erg-3 locus as a mutagenesis target. using this assay we screened 132 wild-isolated neurospora crassa strains for ability to dominantly suppress rip. rip was exceptionally inefficient in crosses with the wild isolates sugartown (p0854) and adiopodoume-7 (p4305), thereby suggesting the presence of dominant rip suppressors in these strains. in other experiments, we found no evidence for dominant rip suppressi ...200111910125
fertile hypomorphic argonaute (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.transgene-induced post-transcriptional gene silencing (ptgs) results from specific degradation of rnas that are homologous with the transgene transcribed sequence. this phenomenon, also known as cosuppression in plants and quelling in fungi, resembles rna interference (rnai) in animals. indeed, cosuppression/quelling/rnai require related paz/piwi proteins (ago1/qde-2/rde-1), indicating that these mechanisms are related. unlike neurospora crassa qde-2 and caenorhabditis elegans rde-1 mutants, whi ...200211910010
control of cpnpg dna methylation by the kryptonite histone h3 methyltransferase.gene silencing in eukaryotes is associated with the formation of heterochromatin, a complex of proteins and dna that block transcription. heterochromatin is characterized by the methylation of cytosine nucleotides of the dna, the methylation of histone h3 at lysine 9 (h3 lys 9), and the specific binding of heterochromatin protein 1 (hp1) to methylated h3 lys 9 (refs 1-7). although the relationship between these chromatin modifications is generally unknown, in the fungus neurospora crassa, dna me ...200211898023
the reverse transcriptase of the r2 non-ltr retrotransposon: continuous synthesis of cdna on non-continuous rna templates.r2 is a non-long terminal repeat (non-ltr) retrotransposon that inserts into the 28 s rrna genes of arthropods. the element encodes two enzymatic activities: an endonuclease that specifically cleaves the 28 s gene target site, and a reverse transcriptase (rt) that can use the 3' end of the cleaved dna to prime reverse transcription. r2 rt only utilizes rna templates that contain the 3' untranslated region of the r2 element as templates in this target primed reverse transcription (tprt) reaction. ...200211866511
molecular dissection of alleles of the osmotic-1 locus of neurospora crassa.two-component histidine kinases of bacteria, plants, and fungi are involved in the regulation of intracellular events in response to changes in external environmental conditions. fungal histidine kinases play important roles in osmoregulation, in vivo and in planta virulence, and sensitivity to certain classes of antifungals. the osmotic-1 (os-1) locus of neurospora crassa encodes a predicted protein with homology to histidine kinases and appears to be an osmosensor. mutants of the os-1 locus ar ...200211848677
the opposed effect of 5-azacytidine and light on the development of reproductive structures in neurospora crassa.blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of neurospora crassa. the dna methylation inhibitor, 5-azacytidine (3-300 microm), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. the addition of 300 microm 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by abou ...200211841042
the sterol c-14 reductase encoded by the neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis.sterol c-14 reductase catalyses the reduction of the delta(14,15) bond in intermediates in the sterol biosynthesis pathway using nadph as a cofactor. we have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol c-14 reductase from neurospora crassa. the effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding n. crassa mutant ( erg-3). the non-complement ...200211810252
the ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in neurospora crassa.somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. in filamentous fungi, somatic cell fusion occurs during vegetative growth. filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. the molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth ...200211805054
large scale analysis of sequences from neurospora crassa.after 50 years of analysing neurospora crassa genes one by one large scale sequence analysis has increased the number of accessible genes tremendously in the last few years. being the only filamentous fungus for which a comprehensive genomic sequence database is publicly accessible n. crassa serves as the model for this important group of microorganisms. the mips n. crassa database currently holds more than 16 mb of non-redundant data of the chromosomes ii and v analysed by the german neurospora ...200211792448
a pest-like element in frequency determines the length of the circadian period in neurospora crassa.frequency (frq) is a crucial element of the circadian clock in neurospora crassa. in the course of a circadian day frq is successively phosphorylated and degraded. here we report that two pest-like elements in frq, pest-1 and pest-2, are phosphorylated in vitro by recombinant ck-1a and ck-1b, two newly identified neurospora homologs of casein kinase 1 epsilon. ck-1a is localized in the cytosol and the nuclei of neurospora and it is in a complex with frq in vivo. deletion of pest-1 results in hyp ...200111742984
epigenetic crosstalk.in the november 15 issue of nature, report that disruption of histone methylation in the fungus neurospora crassa results in the elimination of dna methylation. this demonstrates that chromatin structure can pattern dna methylation and suggests that different epigenetic layers engage in complex crosstalk.200111741529
mutations in subunit c of the vacuolar atpase confer resistance to bafilomycin and identify a conserved antibiotic binding site.bafilomycin a1, a potent inhibitor of vacuolar h(+)-atpases (v-atpase), inhibited growth of neurospora crassa in medium adjusted to alkaline ph. ninety-eight mutant strains were selected for growth on medium (ph 7.2) containing 0.3 or 1.0 microm bafilomycin. three criteria suggested that 11 mutant strains were altered in the v-atpase: 1) these strains accumulated high amounts of arginine when grown at ph 5.8 in the presence of bafilomycin, 2) the mutation mapped to the locus of vma-3, which enco ...200211724795
a histone h3 methyltransferase controls dna methylation in neurospora crassa.dna methylation is involved in epigenetic processes such as x-chromosome inactivation, imprinting and silencing of transposons. we have demonstrated previously that dim-2 encodes a dna methyltransferase that is responsible for all known cytosine methylation in neurospora crassa. here we report that another neurospora gene, dim-5, is required for dna methylation, as well as for normal growth and full fertility. we mapped dim-5 and identified it by transformation with a candidate gene. the mutant ...200111713521
the involvement of photoregulated dna methylation into a choice between sexual and asexual developmental pathways in neurospora crassa. 201311712181
genetic interactions between clock mutations in neurospora crassa: can they help us to understand complexity?recent work on circadian clocks in neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. however, a number of other genes are known that affect either the period or temperature compensation of the rhythm. these include the period (no relationship to the period gene of drosophila) genes and a number of genes that affect cellular metabolism. how these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered ...200111710978
rhythms of differentiation and diacylglycerol in neurospora.although the fungus neurospora crassa is a relatively simple lower eukaryote, its circadian system may be more complex than previously thought. in this paper we review evidence suggesting that there may be several output pathways coupled in complex ways to a single oscillator, or that there may be more than one oscillator driving independent output pathways. we have described two new rhythms in neurospora that are not tightly coupled to the rhythm of conidiation bands that is the standard assay ...200111710977
the neurospora circadian clock: simple or complex?the fungus neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. in this review we concentrate on recent work relating to the complexity of the circadian system in this organism. we discuss: the advantages of neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for ...200111710976
structure of a fast kinesin: implications for atpase mechanism and interactions with microtubules.we determined the crystal structure of the motor domain of the fast fungal kinesin from neurospora crassa (nckin). the structure has several unique features. (i) loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. the displacement in switch 1 resembles that of the g-protein ra ...200111707393
new (and used) approaches to the study of fungal pathogenicity.the fungi are the most economically important plant pathogens and continue to be the focus of extensive research with a wide variety of methodologies. enhancements in microscopy techniques have increased our ability to visualize the intimate interaction of fungi and their host plants. improving methods allow pharmacological inhibition and genetic dissection of the determinants of fungal pathogenicity in a gene-by-gene approach. identification and analysis of genes differentially transcribed in w ...200111701869
on complex i and other nadh:ubiquinone reductases of neurospora crassa mitochondria.the mitochondrial complex i is the first component of the respiratory chain coupling electron transfer from nadh to ubiquinone to proton translocation across the inner membrane of the organelle. the enzyme from the fungus neurospora crassa is similar to that of other organisms in terms of protein and prosthetic group composition, structure, and function. it contains a high number of polypeptide subunits of dual genetic origin. most of its subunits were cloned, including those binding redox group ...200111695829
an analysis of gene-finding programs for neurospora crassa.computational gene identification plays an important role in genome projects. the approaches used in gene identification programs are often tuned to one particular organism, and accuracy for one organism or class of organism does not necessarily translate to accurate predictions for other organisms. in this paper we evaluate five computer programs on their ability to locate coding regions and to predict gene structure in neurospora crassa. one of these programs (ffg) was designed specifically fo ...200111673234
recombination events in neurospora crassa may cross a translocation breakpoint by a template-switching mechanism.to assist investigation of the effect of sequence heterology on recombination in neurospora crassa, we inserted the herpes simplex thymidine kinase gene (tk) as an unselected marker on linkage group i, giving a gene order of cen-his-3-tk-cog-lpl. we show here that in crosses heterozygous for tk, conversion of a his-3 allele on one homolog is accompanied by transfer of the heterologous sequence between cog and his-3 from the other homolog, indicating that recombination is initiated centromere-dis ...200111606534
epistatic and synergistic interactions between circadian clock mutations in neurospora crassa.we identified a series of epistatic and synergistic interactions among the circadian clock mutations of neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. the period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. the prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is consider ...200111606531
dynactin-membrane interaction is regulated by the c-terminal domains of p150(glued).dynactin has been proposed to link the microtubule-associated motor cytoplasmic dynein with membranous cargo; however, the mechanism by which dynactin-membrane interaction is regulated is unknown. here we show that dynein and dynactin exist in discrete cytosolic and membrane-bound states in the filamentous fungus neurospora crassa. results from in vitro membrane-binding studies show that dynein and dynactin-membrane interaction is co-dependent. p150(glued) of dynactin has been shown to interact ...200111571270
the neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) gene.the circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. very little is known about the regulation of outputs from the clock. in neurospora crassa, the circadian clock tr ...200111532152
modeling circadian oscillations with interlocking positive and negative feedback loops.both positive and negative feedback loops of transcriptional regulation have been proposed to be important for the generation of circadian rhythms. to test the sufficiency of the proposed mechanisms, two differential equation-based models were constructed to describe the neurospora crassa and drosophila melanogaster circadian oscillators. in the model of the neurospora oscillator, frq suppresses frq transcription by binding to a complex of the transcriptional activators wc-1 and wc-2, thus yield ...200111517254
seasonality and photoperiodism in fungi.this review gives a retrospective of what is known about photoperiodism in fungi, which is largely based on reports about seasonal spore concentrations. relatively few species have been investigated under laboratory conditions, so that our knowledge whether seasonal reproduction in fungi is mainly a direct response to environmental conditions or whether it involves a photoperiodic machinery with memory capacities and a relationship to the circadian system is extremely limited. to form a basis fo ...200111506384
potassium gradients in the growing hyphae of neurospora crassa.x-ray electron probe microanalysis of a neurospora crassa hyphal preparation revealed a significant heterogeneity of the potassium content in the hyphae. low-potassium areas were found in a close proximity of the growing tip and in the distal part of the hyphae in the submembrane area of about 1 microm width. the intracellular potassium concentration was shown to change more than 3-5 times from apical to distal regions of the leading hyphae, the potassium contents in the apical part of the hypha ...200111497103
regulation of the tip-high [ca2+] gradient in growing hyphae of the fungus neurospora crassa.previous work has shown that hyphal elongation in the fungus neurospora crassa requires a tip-high cytosolic ca2+ gradient. the source of the ca2+ appears to be intracellular stores as there is no net transplasma membrane ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. to decode the internal mechanisms which generate and maintain the tip-high ca2+ gradient we first identified calcium regulators ...200111484929
dim-2 encodes a dna methyltransferase responsible for all known cytosine methylation in neurospora.to understand better the control of dna methylation, we cloned and characterized the dim-2 gene of neurospora crassa, the only eukaryotic gene currently known in which mutations appear to eliminate dna methylation. the dim-2 gene is responsible for methylation in both symmetrical and asymmetrical sites. we mapped dim-2 between wc-1 and un-10 on linkage group (lg) viir and identified the gene by rflp mapping and genetic complementation. dim-2 encodes a 1454 amino acid protein including a c-termin ...200111483533
chloride channel antagonists perturb growth and morphology of neurospora crassa.the chloride channel antagonists anthracene-9-carboxylic acid, ethacrynic acid and niflumic acid were found to be fungistatic and morphogenic when tested against the ascomycete neurospora crassa. potency increased with decreasing ph, suggesting that the protonated forms of the compounds were active. niflumic acid produced the most pronounced growth aberrations which may reflect an ability to acidify the cytoplasm and block the plasma membrane anion channel of n. crassa.200111470368
a fungus among us: the neurospora crassa circadian system.neurospora crassa is the only molecular genetic model system for circadian rhythms research in the fungi. its strengths as a model organism lie in its relative simplicity--compared to photosynthesizing and vertebrate organisms, it is a stripped-down version of life. it forms syncitial hyphae, propagates and reproduces, and the circadian clock is manifest in numerous processes therein. as with other model circadian systems, neurospora features a transcription/translation feedback loop that is fun ...200111463212
null mutants of the neurospora actin-related protein 1 pointed-end complex show distinct phenotypes.dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. actin-related protein 1 (arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. an arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and arp11 subunits. the functional roles of these subunits have not been determined. recently, we reported the cloning of an apparent ho ...200111452013
[effect of inhibitors of enzymatic dna methylation on the formation of reproductive structures and carotenoid production in neurospora crassa].the effect of inhibitors of dna methylation on light-sensitive developmental stages of the filamentous fungus neurospora crassa was studied. under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microm) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). after treatment of the mycelium with 5-azacytidine, the protoperithecia formation was acco ...201311443894
elimination of active tad elements during the sexual phase of the neurospora crassa life cycle.tad is an active line-like retrotransposon isolated from the adiopodoumé strain of neurospora crassa. extensive analysis of other neurospora strains has revealed no other strain with active tad, but all strains tested have multiple copies of defective tad elements. we have examined the ability of tad to survive during the sexual cycle of neurospora and find that active tad is rapidly eliminated. the characteristics of this elimination suggest that the repeat-induced point mutation (rip) mechanis ...200111407885
characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of neurospora crassa.osmotic-sensitive (os-1) mutant alleles in neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. we have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type i, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type ii, which are highly sensitive to osmotic stress but moderately resistant to fungicides. to explai ...200111374161
mobilisation of trehalose in mutants of the cyclic amp signalling pathway, cr-1 (crisp-1) and mcb (microcycle conidiation), of neurospora crassa.the influence of the camp-signalling pathway on the metabolism of trehalose in neurospora crassa was investigated. the changes in intracellular trehalose concentration were measured in two mutants affected in components of the camp-signalling pathway: cr-1 (crisp-1), deficient in adenylyl cyclase activity, and mcb (microcyclic conidiation), deficient in the regulatory subunit of pka. rapid mobilisation of intracellular trehalose in the wild-type occurred, either at the onset of germination, or a ...200111356572
vvd is required for light adaptation of conidiation-specific genes of neurospora crassa, but not circadian conidiation.con-10 and con-6 are two of the conidiation (con) genes of neurospora crassa that were identified based on their preferential expression during macroconidiophore development. they are also regulated by several other environmental stimuli independent of development, including a transient induction by light. we identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to ligh ...200111343403
interaction of the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) with the group i intron p4-p6 domain. thermodynamic analysis and the role of metal ions.the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) functions in splicing group i introns by promoting the formation of the catalytically active structure of the intron's catalytic core. previous studies suggested a model in which the protein binds first to the intron's p4-p6 domain, and then makes additional contacts with the p3-p9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. here, we analyzed the interaction ...200111327760
electron tomography of mitochondria after the arrest of protein import associated with tom19 depletion.in a mutant form of neurospora crassa, in which sheltered rip (repeat induced point mutation) was used to deplete tom19, protein transport through the tom/tim pathway is arrested by the addition of p-fluorophenylalanine (fpa). using intermediate-voltage electron tomography, we have generated three-dimensional reconstructions of 28 fpa-treated mitochondria at four time points (0-32 h) after the addition of fpa. we determined that the cristae surface area and volume were lost in a roughly linear m ...200111302518
escape from repeat-induced point mutation of a gene-sized duplication in neurospora crassa crosses that are heterozygous for a larger chromosome segment duplication.in neurospora crassa the ability of an ectopic gene-sized duplication to induce repeat-induced point mutation (rip) in its target gene was suppressed in crosses that were heterozygous for another larger chromosome segment duplication. specifically, the frequency of rip in the erg-3 gene due to a 1.3-kb duplication was reduced if the chromosome segment duplications dp(iiir > [i;ii]) ar17, dp(vir > iiir) oy329, or dp(ivr > vii) s1229 were present in either the same or the other parental nucleus of ...200111290714
a point mutation in nucleoside diphosphate kinase results in a deficient light response for perithecial polarity in neurospora crassa.in neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (ndk)-1 is rapidly enhanced after blue light irradiation. we have investigated the function of ndk-1 in the blue light signal transduction pathway. a mutant called psp (phosphorylation of small protein) shows undetectable phosphorylation of ndk-1 and is defective in light-responsive regulation of perithecial polarity. sequencing analysis of ndk-1 cdna by reverse transcription-polymerase chain reaction revealed that prolin ...200111287415
tests of a cellular model for constant branch distribution in the filamentous fungus neurospora crassa.the growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. in neurospora crassa, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by n. crassa has not been proposed. we de ...200111282634
hex-1, a gene unique to filamentous fungi, encodes the major protein of the woronin body and functions as a plug for septal pores.we have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or woronin bodies, of neurospora crassa. analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. when grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. the hex-1 protein was predicted to be 19,125 da. analysis of the n- ...200011273682
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