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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
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
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
carbon source affects pka-dependent polarity of neurospora crassa in a cre-1-dependent and independent manner.a defect in mcb, encoding the camp-dependent protein kinase a (pka) regulatory subunit in neurospora crassa, which confers an apolar growth phenotype, is accompanied by an increase in pka activity levels. both pka and cre-1 [a key carbon catabolite repression (ccr) regulator] mediate the cellular response to carbon-source availability. inactivation of the cre-1 gene resulted in reduced growth rate, abnormal hyphal morphology and altered ccr. both pka and cre-1 affected morphology in a carbon-dep ...200817625933
[a study of the nitrate and nitrite discharge from the mutant cells of neurospora crassa lacking nitrate and nitrite reductase activities].the neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. for nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. unlike nit-2, the content of nitrite in the cultivation medium of ...201317619581
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
efficient recursive linking algorithm for computing the likelihood of an order of a large number of genetic markers.assuming no interference, a multi-locus genetic likelihood is implemented based on a mathematical model of the recombination process in meiosis that accounts for events up to double crossovers in the genetic interval for any specified order of genetic markers. the mathematical model is realized with a straightforward algorithm that implements the likelihood computation process. the time complexity of the straightforward algorithm is exponential without bound in the number of genetic markers and ...200617369637
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
structural and kinetic alterations of constitutive conidial alkaline phosphatase from the osmotically-sensitive mutant of neurospora crassa.the osmotically-sensitive os-1 mutant of neurospora crassa overproduced conidial alkaline phosphatase. the enzyme was purified by phenyl-sepharose cl-4b chromatography and sephadex g-200 gel filtration. page analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. the enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kda. two protein bands (36 and 62 kda) were observed in sds-page, suggesting that the native enzyme ...200617176763
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
identification of non-specific alkaline phosphatases in hyphal cells of the fungus neurospora crassa by in situ histochemistry.the present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either mg(2+) or edta. another alkaline phosphatase activity, whose location in the hyphal cell is regulated by pho ...200617117363
the rhythms of life: circadian output pathways in neurospora.research in neurospora crassa pioneered the isolation of clock-controlled genes (ccgs), and more than 180 ccgs have been identified that function in various aspects of the fungal life cycle. many clock-controlled genes are associated with damage repair, stress responses, intermediary metabolism, protein synthesis, and development. the expression of most of these genes peaks just before dawn and appears to prepare the cells for the desiccation, mutagenesis, and stress caused by sunlight. progress ...200617107934
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
[oxidative stress and cell differentiation in neurospora crassa].environmental stress factors induce oxidative stress in fungi by increasing the intracellular concentrations of reactive oxygen species (ros). in the mycelium, ros act as signal molecules needed for cytodifferentiation at certain stages of the development of fungi. generation of ros in cells induces the activation of antioxidant protective mechanisms. the purpose of this communication is to analyze the role of ros in light signal transduction, mediated in neurospora crassa cells by the white col ...200617025175
lithium leads to an increased frq protein stability and to a partial loss of temperature compensation in the neurospora circadian clock.in many organisms, the presence of lithium leads to an increase of the circadian period length. in neurospora crassa, it was earlier found that lithium results in a decrease of overall growth and increased circadian periods. in this article, the authors show that lithium leads to a reduction of frq degradation with elevated frq levels and to a partial loss of temperature compensation. at a concentration of 13 mm lithium, frq degradation is reduced by about 60% while, surprisingly, the activity o ...200616998153
proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in neurospora crassa.to investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (cdp ii). immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a sing ...200616972126
karyotyping of neurospora crassa using synaptonemal complex spreads of translocation quadrivalents.the purposes of the present research are (i) to establish the karyotype of neursopora crassa using visualization of kinetochores in the synaptonemal complex (sc) spreads, (ii) to assign each chromosome to a linkage group, and (iii) to examine chromosome pairing and recombination nodules in quadrivalents. two strains containing reciprocal translocations were used: t(i;ii)4637, which involves linkage groups i and ii, and alcoy, which contains 3 independent translocations involving i and ii, iv and ...200616936840
circadian rhythms in neurospora crassa and other filamentous fungi. 200616896204
[a comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type neurospora crassa strain and mutants, white collar-1 and white collar-2].a comparative study of the changes in the components of the antioxidant defense system (ads), the activity of superoxide dismutase (sod) and catalase and the level of extractable sh-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) neurospora crassa strains was performed. oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable sh-groups an ...200616878551
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
functional analysis of an endo-1,6-beta-d-glucanase gene (neg-1) from neurospora crassa.the 1,6-beta-d-glucanase gene (neg1) of neurospora crassa was disrupted by repeat-induced point mutations. sequence analysis of the neg1 gene in the r12-1 mutant showed that 9 nucleotides within the coding region of the gene changed from gc to at. the base transition of c to a at position 662 resulted in a codon. no apparent phenotypic changes were observed in the mutant, but congo-red, sds, and cetyltrimethyl ammonium bromide (ctab), which affect fungal cell walls or membranes, markedly inhibit ...200616861813
light-dependent subcellular localization of nucleoside diphosphate kinase-1 in neurospora crassa.nucleoside diphosphate kinase (ndk) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. in neurospora crassa, ndk-1 is suggested to control catalases in response to heat, oxidative stress and light. in this study, we identified the presence of ndk-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fr ...200616842360
the ste20/germinal center kinase pod6 interacts with the ndr kinase cot1 and is involved in polar tip extension in neurospora crassa.members of the ste20 and ndr protein kinase families are important for normal cell differentiation and morphogenesis in various organisms. we characterized pod6 (ncu02537.2), a novel member of the gck family of ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in the filamentous fungus neurospora crassa. pod-6 and the ndr kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered ...200616822837
light regulation of asexual development in the rice blast fungus, magnaporthe oryzae.light is a major environmental factor that influences many biological processes. we characterized the roles of light in asexual development (including the formation of aerial hyphae and conidiophore) in magnaporthe oryzae, which is the causal agent of rice blast disease. our data revealed a complex nature of light regulation in the asexual developments of m. oryzae. asexual development of m. oryzae is suppressed by blue light in a light/dark cycling environment and asexual spore release is contr ...200616765070
chromosome pairing and meiotic recombination in neurospora crassa spo11 mutants.some organisms, such as mammals, green plants and fungi, require double-strand breaks in dna (dsbs) for synapsis of homologous chromosomes at pachynema. drosophila melanogaster and caenorhabditis elegans are exceptions, achieving synapsis independently of dsb. spo11 is responsible for generating dsbs and perhaps for the initiation of recombination in all organisms. although it was previously suggested that neurospora may not require dsbs for synapsis, we report here that mutation of neurospora s ...200616758206
a nonself recognition gene complex in neurospora crassa.nonself recognition is exemplified in the fungal kingdom by the regulation of cell fusion events between genetically different individuals (heterokaryosis). the het-6 locus is one of approximately 10 loci that control heterokaryon incompatibility during vegetative growth of n. crassa. previously, it was found that het-6-associated incompatibility in oak ridge (or) strains involves two contiguous genes, het-6 and un-24. the or allele of either gene causes "strong" incompatibility (cell death) whe ...200616751666
two circadian timing circuits in neurospora crassa cells share components and regulate distinct rhythmic processes.in neurospora crassa, frq, wc-1, and wc-2 proteins comprise the core circadian frq-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. however, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of frq (collectively termed frq-less oscillators [flos]). whether or not these represent temperature-compensated, entrainable circadian oscillators is ...200616731655
flexibility of the neck domain enhances kinesin-1 motility under load.kinesin-1 is a dimeric motor protein that moves stepwise along microtubules. a two-stranded alpha-helical coiled-coil formed by the neck domain links the two heads of the molecule, and forces the motor heads to alternate. by exchanging the particularly soft neck region of the conventional kinesin from the fungus neurospora crassa with an artificial, highly stable coiled-coil we investigated how this domain affects motor kinetics and motility. under unloaded standard conditions, both motor constr ...200616714343
characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in neurospora.in continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (cdre) in the -3730bp upstream region of copper-induced metallothionein (cumt) gene of neurospora [k.s. kumar, s. dayananda, c. subramanyam, copper alone, but not oxidative stress, induces copper-metallothionein gene in neurospora crassa, fems microbiol. lett. 242 (2005) 45-50], we isolated and characterized the cdre-binding protein. the cloned upstream region of cumt gene was used ...200616707100
how temperature affects the circadian clock of neurospora crassa.light and temperature are major environmental cues that influence circadian clocks. the molecular effects of these zeitgebers on the circadian clock of neurospora crassa have been studied intensively during the last decade. while signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. here we summarize briefly the current knowledge about the effects of temperature on t ...200616687282
entrainment of the neurospora circadian clock.neurospora crassa has been systematically investigated for circadian entrainment behavior. many aspects of synchronization can be investigated in this simple, cellular system, ranging from systematic entrainment and drivenness to masking. clock gene expression during entrainment and entrainment without clock genes suggest that the known transcription/translation feedback loop is not alone responsible for entrainment in neurospora.200616687281
biochemical properties of a thermostable phytase from neurospora crassa.a gene (ncphy) encoding a putative phytase in neurospora crassa was cloned and expressed in pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. the recombinant phytase (rncphy) hydrolyzed phytic acid with a specific activity of 125 u mg-1, km of 228 micromol l-1, vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees c and a ph optimum of 5.5 and a second ph optimum of 3.5. the enz ...200616630256
microtubule dynamics and organization during hyphal growth and branching in neurospora crassa.by confocal microscopy, we analyzed microtubule (mt) behavior during hyphal growth and branching in a neurospora crassa strain whose mts had been tagged with gfp. images were assembled spatially and temporally to better understand the 3-d organization of the microtubular cytoskeleton and a clearer view of its dynamics. cytoplasmic mts were mainly arranged longitudinally along the hyphal tube. straight segments were rare; most mts showed a distinct helical curvature with a long pitch and a tenden ...200616621627
developmental expression of two forms of arginase in neurospora crassa.n. crassa has two forms of arginase. the physiological role of multiple arginases is not understood. the two forms were shown to be differentially expressed from a single locus (aga) and both proteins are localized to the cytoplasm. the 36-kda protein was expressed in minimal and arginine supplemented medium, whereas the 41-kda form was detected only in the presence of arginine. in this study we examined developmental expression of the two arginase transcripts and proteins in conidia and during ...200616574329
nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in neurospora crassa.nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. when individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. in neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. this domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of h ...200616554411
circadian clock genes frequency and white collar-1 are not essential for entrainment to temperature cycles in neurospora crassa.the fungus neurospora crassa is a model system for investigating the mechanism of circadian rhythmicity, and the core of its circadian oscillator is thought to be a transcription/translation feedback loop involving the products of the frq (frequency), wc-1 (white-collar-1) and wc-2 (white-collar-2) genes. several reports of rhythmicity in frq and wc null mutants have raised questions about how central the frq/wc loop is to the circadian system of neurospora. several research groups have attempte ...200616537415
mutational analysis of the glycosylphosphatidylinositol (gpi) anchor pathway demonstrates that gpi-anchored proteins are required for cell wall biogenesis and normal hyphal growth in neurospora crassa.using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (gpi) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus neurospora crassa. mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the n. crassa gpi anchor biosynthetic pathway, have been characterized. gpi anchor mutants exhibit colonial morphologies, significantly reduced rates ...200616524913
pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in neurospora crassa.neurospora crassa is a self-sterile filamentous fungus with two mating types, mat a and mat a. its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat a cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mrna for the pheromone mfa-1 and the pre-2 receptor; mfa-1 and ccg-4 are the predicted ligands for pre-1 and pre-2, respectively. ...200616524909
[analysis of the secreted proteins encoded by genes in genome of filamental fungus (neurospora crassa)].the completed genome sequence of filamental fungus neurospora crassa has completed and 10,082 open reading frames (orfs) were predicted in whole genome. among them, 437 proteins were secreted ones based on the combination of prediction algorithms signalp v3.0 and psort, transmembrane domains prediction algorithms tmhmm v2.0 and thumbup, potential gpi-anchor sites prediction algorithm big-pi predictor and the subcellular protein location prediction algorithm targetp v1.01. the minimum orf length ...200616520317
deletion variants of neurospora mitochondrial porin: electrophysiological and spectroscopic analysis.mitochondrial porins are predicted to traverse the outer membrane as a series of beta-strands, but the precise structure of the resulting beta-barrel has remained elusive. toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted beta-strands of the neurospora crassa porin. overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of beta ...200616500966
microarray and real-time pcr analyses reveal mating type-dependent gene expression in a homothallic fungus.sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. to analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene smta-1 encoding a high-mobility group domain (hmg) protein. the deltasmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. to identify genes expressed u ...200616482473
construction of pdestr, a gateway vector for gene disruption in filamentous fungi.we have constructed pdestr, a destination vector of gateway system especially for gene targeting and disruption in filamentous fungi. the vector was constructed by removing the multicloning site of pgem-t easy vector, and inserting hygromycin phosphotransferase gene construct from pcb1004, and a gateway vector conversion cassette. in order to construct a dna for gene disruption, only an inverse-polymerase chain reaction (pcr) amplification of the restricted, target sequence is needed. after the ...200616479358
cell-wall-bound metal ions are not taken up in neurospora crassa.to establish the relevance of the cell wall in metal ion transport, cobalt uptake was examined in neurospora crassa. cobalt taken up was largely surface bound (>90%), resulting in a release of calcium and magnesium. surface-bound cobalt could not enter intracellular locations upon further incubation of mycelia in a metal-free medium. saturation of the surface with one metal augured subsequent dose-dependent entry of a different metal into intracellular locations. in comparison with the cobalt-re ...200516462860
mannosyltransferase is required for cell wall biosynthesis, morphology and control of asexual development in neurospora crassa.two neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. the relevant mutations were mapped to the same locus on the sequenced neurospora genome. the mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with pcr amplification and a sequencing assay. the results demon ...200616457356
homology effects in neurospora crassa.it has become clear in the past few years that eukaryotic organisms possess different genetic systems to counter viruses, transposons and other repeated elements such as transgenes that could otherwise accumulate in the genome. in addition to serving as a model organism for genetic, biochemical and molecular studies, neurospora crassa has proved to be a paradigm for the study of gene-silencing mechanisms. indeed, its genome can be protected from expansion of selfish nucleic acids by a variety of ...200616445744
asexual transmission, non-suppressiveness and meiotic extinction of small plasmid-like derivatives of the mitochondrial dna in neurospora crassa.for reasons that are not obvious, sets of related plasmid-like elements that consist of short segments of dna that overlap the 5' terminal region of the mitochondrial large-subunit rrna gene sometimes appear spontaneously and become amplified in the mitochondria of some cytochrome-deficient and/or uv-sensitive mutants of neurospora crassa. these elements are transmitted efficiently through hyphal anastomoses and appear to invade the mitochondria of recipient strains, but they do not cause senesc ...200616386438
biogenesis and replication of small plasmid-like derivatives of the mitochondrial dna in neurospora crassa.for reasons that are not obvious, sets of related, small, plasmid-like elements appear spontaneously and become amplified in the mitochondria of some cytochrome-deficient and/or uv-sensitive mutants of neurospora crassa. these plasmid-like dnas are multimeric series of circular molecules, each consisting of a finite number of identical tandem repeats of a relatively short mtdna-derived nucleotide sequence (monomer). the plasmid-like elements that have been characterized in this study consist of ...200616386436
chromosome segment duplications in neurospora crassa and their effects on repeat-induced point mutation and meiotic silencing by unpaired dna.the size and extent of four neurospora crassa duplications, dp(ar17), dp(ibj5), dp(oy329), and dp(b362i), was determined by testing the coverage of rflp markers. the first three duplications were all > approximately 350 kb and have been shown in earlier studies to act as dominant suppressors of repeat-induced point mutation (rip) in gene-sized duplications, possibly via titration of the rip machinery. dp(b362i), which is only approximately 117 kb long, failed to suppress rip. rip suppression in ...200616361235
structural analysis of the zen-4/cemklp1 motor domain and its interaction with microtubules.the centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. it is composed of two copies each of the kinesin-like protein zen-4, a caenorhabditis elegans mklp-1 (kinesin-6 family), and the rhogap cyk-4. by using cryo-electron microscopy and helical 3d reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of zen-4 and microtub ...200616361109
laser microsurgery permits fungal plasma membrane single-ion-channel resolution at the hyphal tip.a method for formation of high-electrical-resistance seals on the neurospora crassa plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. cell wall reformation is delayed by brefeldin. this method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth.199816349556
the relationship between frq-protein stability and temperature compensation in the neurospora circadian clock.temperature compensation is an important property of all biological clocks. in neurospora crassa, negative-feedback regulation on the frequency (frq) gene's transcription by the frq protein plays a central role in the organism's circadian pacemaker. earlier model calculations predicted that the stability of frq should determine the period length of neurospora's circadian rhythm as well as the rhythm's temperature compensation. here, we report experimental frq protein stabilities in frq mutants a ...200516314576
bioclonedb: a database application to manage dna sequence and gene expression data.bioclonedb is a user-friendly database with a web interface to assist molecular genetics laboratories in managing a local repository of sequence information linked to dna clones. this tool is designed to assist in high-throughput sequence and gene expression projects, providing a link between both types of information. the unique feature of the application is the automation of batch sequence annotation following blast((r)) searches, which is supported by easy-to-use web interfaces. furthermore, ...200516309345
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