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improving the efficiency of gene replacements in neurospora crassa: a first step towards a large-scale functional genomics project.here, we report the use of the mating type heterokaryon incompatibility system as a counterselection to increase the probability of identifying gene replacements in neurospora crassa. we compared the frequencies of gene replacements observed among transformants obtained by using plasmids with or without the mat a-1(+) gene (hereby called "toxic gene") placed adjacent to disruption cassettes. on an average, we were 20x more likely to identify a correct gene replacement by incorporating the toxic ...200212223190
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
repeat-induced point mutation (rip) in magnaporthe grisea: implications for its sexual cycle in the natural field context.repeat-induced point mutation (rip) is a process that detects dna duplications and peppers their sequences with c:g to t:a transitions in the sexual phase of the life cycle. so far, this unique mechanism has been identified as a currently active process in only two fungal species, neurospora crassa and podospora anserina. to determine whether a rip-like process operates in the plant pathogenic fungus magnaporthe grisea, the retrotransposon maggy and the hygromycin b phosphotransferase gene were ...200212207702
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
[effect of microbial elicitors on sclerotia biomass and carotenoid yield in surface cultures of penicillium sp.pt95].six kinds of elicitors were prepared respectively from neurospora crassa, monascus purpureus, sporobolomyces roseus, rhodotorula rubra, nocardia sp. n89 and actinoplanes sp. a05. when penicillium sp. pt95 was incubated in czapek's agar plates containing appropriate amounts of elicitors, both its sclerotia biomass and carotenoid content accumulated in sclerotia were enhanced significantly (p < 0.01). among tested elicitors, the elicitors from the fungi n. crassa, m. purpureus, s.-roseus and r. ru ...200212192877
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
cloning and expression of an endo-1,6-beta-d-glucanase gene (neg1) from neurospora crassa.a gene (neg1) encoding an endo-1,6-beta-d-glucanase from neurospora crassa was cloned. the putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-d-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. the purified recombinant protein (neg1) obtained from escherichia coli showed 1,6-beta-d-glucanase activity. no genes similar in sequence were found in yeasts and fungi.200212162562
regulation and oxidation of two large monofunctional catalases.the two neurospora crassa catalase genes cat-1 and cat-3 were shown to encode cat-1 and cat-3 large monofunctional catalases. cat-1 and cat-3 genes are regulated differentially during the asexual life cycle and under stress conditions. a stepwise increase in catalase activity occurs during conidiation. conidia have 60 times more catalase activity than exponentially growing hyphae. cat-1 activity was predominant in conidia, during germination and early exponential growth. it was induced during pr ...200212160934
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
cellular responses to environmental salinity in the halophilic black yeast hortaea werneckii.the development of crop plants with increased salt tolerance necessitates the study of naturally salt-tolerant eukaryotic species. we studied the bio-synthesis of glycerol as a compatible solute in the halophilic eukaryotic microorganism, black yeast hortaea werneckii. a restriction fragment-differential display technique was used to investigate the transcriptome of the organism. eight differentially expressed genes were identified in response to growth at different salinities. although the puta ...200212139614
cloning and characterization of the gene encoding ribosomal p0 phosphoprotein from neurospora crassa.a gene for ribosomal protein p0 that belongs to the family of ribosomal p proteins was isolated from a neurospora crassa cdna library, using polyclonal antibodies against recombinant p0 protein from saccharomyces cerevisiae. this is the first gene for ribosomal p0 protein to be cloned from filamentous fungi. the derived p0 protein sequence has a strong homology to other eukaryotic p0 proteins; yet, there is a notable alteration in the conservative c-terminal region, placing this protein among th ...200212136931
an osmosensing histidine kinase mediates dicarboximide fungicide resistance in botryotinia fuckeliana (botrytis cinerea).a two-component histidine protein kinase gene, homologous to os-1 from neurospora crassa, was cloned and sequenced from a single ascospore isolate of botryotinia fuckeliana. a series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. genetic crosses of the mutants with an authentic daf1 strain showed that the phenotypes mapped to this locus. single point mutations (seven t ...200212135574
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
a regulator gene for acetate utilisation from neurospora crassa.the neurospora crassa homologue of the aspergillus nidulans regulatory gene facb has been cloned. the gene encodes a putative transcriptional activator of 865 amino acids that contains a dna-binding domain with a zn(ii)(2)cys(6) binuclear cluster, a linker region and a leucine zipper-like heptad repeat. two internal amino acid sequences are identical to peptide sequences determined from proteolytic fragments of a dna-binding protein complex specific for genes involved in acetate utilisation and ...200212111557
interaction between mating-type proteins from the homothallic fungus sordaria macrospora.mating-type genes control sexual development in ascomycetes. little is known about their function in homothallic species, which are self-fertile and do not require a mating partner for sexual reproduction. the function of mating-type genes in the homothallic fungus sordaria macrospora was assayed using a yeast system in order to find properties typical of eukaryotic transcription factors. we were able to demonstrate that the mating-type proteins smta-1 and smta-1 have domains capable of activati ...200212111096
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
functional analysis of the c6 zinc finger gene pro1 involved in fungal sexual development.the pro1 gene, controlling fruiting body development in the homothallic ascomycete sordaria macrospora, encodes a c6 zinc finger protein with a typical dna binding domain of gal4-like c6 zinc finger proteins as well as a putative nuclear targeting signal. in the corresponding mutant pro1, the pro1 gene is deleted, and the transition of primordia into mature fruiting bodies is prevented. to further characterize the pro1 polypeptide, the yeast system was used for identifying a transactivation doma ...200212081464
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
the upr-1 gene encodes a catalytic subunit of the dna polymerase zeta which is involved in damage-induced mutagenesis in neurospora crassa.the upr-1 mutant was one of the first mutagen-sensitive mutants to be isolated in neurospora crassa. however, the function of the upr-1 gene has not yet been elucidated, although some genetic and biochemical data have been accumulated. in order to clone the upr-1 gene, we performed a chromosome walk from the mat locus, the closest genetic marker to upr-1 for which a molecular probe was available, towards the centromere, and a chromosomal contig of about 300-400 kb was constructed. some of these ...200212073042
a cytosine methyltransferase homologue is essential for repeat-induced point mutation in neurospora crassa.during sexual development, neurospora crassa inactivates genes in duplicated dna segments by a hypermutation process, repeat-induced point mutation (rip). rip introduces c:g to t:a transition mutations and creates targets for subsequent dna methylation in vegetative tissue. the mechanism of rip and its relationship to dna methylation are not fully understood. mutations in dim-2, a dna methyltransferase (dmt) responsible for all known cytosine methylation in neurospora, does not prevent rip. we u ...200212072568
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
toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mrnas.the ability to map the position of ribosomes and their associated factors on mrnas is critical for an understanding of translation mechanisms. earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. while these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. here we describe an alternative ...200212054887
disruption of iron-sulphur cluster n2 from nadh: ubiquinone oxidoreductase by site-directed mutagenesis.we have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kda subunit of complex i (ec 1.6.5.3) from neurospora crassa, the homologue of the bovine psst polypeptide. mitochondria from mutant nuo19.3 lack the peripheral arm of complex i while its membrane arm accumulates. transformation with wild-type cdna rescues this phenotype and assembly of complex i is restored. to interfere with assembly of a proposed bound iron-sulphur cluster, site-directed muta ...200212049648
identification, cloning and expression of the mouse n-acetylglutamate synthase gene.in ureotelic animals, n-acetylglutamate (nag) is an essential allosteric activator of carbamylphosphate synthetase i (cpsi), the first enzyme in the urea cycle. nag synthase (nags; ec 2.3.1.1) catalyses the formation of nag from glutamate and acetyl-coa in liver and intestinal mitochondria. this enzyme is supposed to regulate ureagenesis by producing variable amounts of nag, thus modulating cpsi activity. moreover, inherited deficiencies in nags have been associated with hyperammonaemia, probabl ...200212049647
a 3d model of the voltage-dependent anion channel (vdac).eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. as opposed to the bacterial porins (a large family of protein whose 3d structure has been determined by x-ray diffraction), the structure of eukaryotic porins (also termed vdacs, voltage-dependent anion-selective channels) is still a matter of debate. we analysed the secondary structure of vdac from the yeast saccharomyces cerevisiae, the fungus neurosp ...200212044860
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
carnitine biosynthesis in neurospora crassa: identification of a cdna coding for epsilon-n-trimethyllysine hydroxylase and its functional expression in saccharomyces cerevisiae.the biosynthesis of l-carnitine in eukaryotic organisms was first elucidated in the ascomycete neurospora crassa. the first step of the pathway is catalysed by epsilon-n-trimethyllysine hydroxylase (tmlh), which converts epsilon-n-trimethyllysine into beta-hydroxy-n-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, fe2+ and oxygen. here we report on the cloning of the n. crassa tmlh cdna and its functional expression in saccharomyces cerevisiae. the tmlh cdna contains an op ...200212023072
[arg-13 is implicated in mitochondrial ornithine transport in neurospora crassa].arg-13 is a leaky mutation involved in arginine metabolism. a tight selection is developed using similar amount of lysine and ornithine replacing other nitrogen source in minimal medium. this selection strongly inhibits the growth of arg-13 under stringent sorbose/glucose condition but allows arg-13 to grow under spot test conditions. as ornithine is build up through mitochondrial ornithine biosynthesis and transport from cytoplasm to mitochondria, arg-13 is combined in genetic crosses with arg- ...199812016957
cloning and characterization of vmaa, the gene encoding a 69-kda catalytic subunit of the vacuolar h+-atpase during alkaline ph mediated growth of aspergillus oryzae.screening of a cdna library constructed under alkaline ph mediated growth of aspergillus oryzae implicated a vacuolar h+-atpase gene (vmaa) as a putative candidate involved in alkaline ph adaptation. a. oryzae vmaa genomic dna extended to 2072 bp including three introns and encoded a protein of 605 amino acids. vmaap was homologous to vma-1p from neurospora crassa (71%), vma1p from saccharomyces cerevisiae (69%) and atp6a2 from human (49%). the vmaa cdna complemented s. cerevisiae v-atpase disru ...200212007818
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
molecular and biochemical characterization of the neurospora crassa glycogen synthase encoded by the gsn cdna.glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. the fungus neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. through a pcr methodology, the gs ...200211976968
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
kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate.the herbicide glyphosate (n-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (epsp) synthase activity of the purified arom multienzyme complex from neurospora crassa. inhibition of the epsp synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with k(i) 1.1 microm, and uncompetitive with respect to shikimate-3-phosphate. the kinetic patterns are consistent with a compulsory order sequential mechanism in which either ...198311968207
interaction of mitochondria with microtubules in the filamentous fungus neurospora crassa.the establishment and maintenance of the 3d structure of eukaryotic cells depends on active transport and positioning of organelles along cytoskeletal elements. the biochemical basis of these processes is only poorly understood. we analysed the interaction of mitochondria with microtubules in the filamentous fungus neurospora crassa. mitochondria were fluorescently labelled by expression of matrix-targeted green fluorescent protein. upon isolation, mitochondria collapsed to round spherical struc ...200211956324
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
effects of radiation during space flight on microorganisms and plants on the biosatellite ii and gemini xi missions.the results of recent experiments with the lysogenic bacteria, escherichia coli and salmonella typhimurium, the bread mold neurospora crassa and the flowering plant tradescantia on the biosatellite ii and gemini xi missions will be summarized. in the lysogenic bacteria experiment (dr. rudolf h.t. mattoni, nus corporation) on the biosatellite ii mission significant effects of space flight were found on both growth rate and the induction of prophage. in that part of the neurospora experiment on bo ...196911949688
ferrochelatase in wild-type and in cytoplasmic mutants of neurospora crassa. 196911947230
cycloheximide resistant amino acid incorporation into mitochondrial protein from neurospora crassa in vivo. 196911947193
specificity of mitochondrial and cytoplasmic ribosomes from neurospora crassa in poly-u dependent cell free systems. 196911947164
aldosugar dehydrogenases from neurospora crassa partial purification and characterization of d-arabinose: nad dehydrogenase. 197311947073
insulin inhibition of membrane-bound adenylate cyclase in neurospora crassa. 197311947065
in vitro translation of mitochondrial dna from neurospora crassa. 197211946701
close correlation between antimycin titer and cytochrome b(t) content in mitochondria of chloramphenicol treated neurospora crassa. 197211946688
oxygenases involved in thymine and thymidine metabolism in neurospora crassa. 197211946494
 in the extracts of quercus pedunculata roots, the activities of 5-dehydroquinate hydro-lyase and shikimate: nadp oxydoreductase are eluted in the same fractions whatever separation procedures are used: chromatography on sephadex g 100, deae cellulose and hydroxylapatite, centrifugation in sucrose density gradient, disc electrophoresis. these results are closely related to those obtained on the aggregate of enzymes involved in the shikimate pathway in neurospora crassa. the molecular weight of th ...197111945772
mitochondrial peptide chain elongation factors from neurospora crassa.two complementary peptide chain elongation factors (g and t) have been isolated from a mitochondrial 100,000 g supernatant. both factors are specific for 70 s ribosomes and can be crossed with t and g factors from e. coli.197011945348
electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of neurospora crassa wild type and mi-1 mutant. 196811945305
genes encoding chimeras of neurospora crassa erg-3 and human tm7sf2 proteins fail to complement neurospora and yeast sterol c-14 reductase mutants.the human gene tm7sf2 encodes a polypeptide (sr-1) with high sequence similarity to sterol c-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. in neurospora and yeast this enzyme is encoded by the erg-3 and erg24 genes respectively. in an effort to demonstrate sterol c-14 reductase activity for sr-1 we constructed six recombinant genes coding for chimeras of the neurospora erg-3 and sr-1 protein sequences and tested them for complementation of the neurospora erg-3 muta ...200211937680
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
cia30 complex i assembly factor: a candidate for human complex i deficiency?the human mitochondrial nadh:ubiquinone oxidoreductase (complex i), the first complex of the oxidative phosphorylation system, is composed of at least 42 subunits. little is known about the assembly process of these subunits into the mature complex. recently, two proteins in neurospora crassa have been found to be involved in the assembly of complex i. these proteins are not constituent parts of the mature complex but are associated with smaller intermediate complexes of the assembly process and ...200211935339
functional analysis of the escherichia coli molybdopterin cofactor biosynthesis protein moea by site-directed mutagenesis.five moea mutants were generated by replacing some conserved amino acids of moea by site-directed mutagenesis. the mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moea mutant escherichia coli jrg97 and in vitro neurospora crassa nit-1 nitrate reductase activity. the replacements asp59alagly60ala, asp259ala, pro298alapro301ala abolished the function of moea in mo-molybdopterin formation and stabilization, reflected in the inability to restore nitrate redu ...200211934270
quelling in neurospora crassa. 200211931228
a genetic map of blumeria graminis based on functional genes, avirulence genes, and molecular markers.a genetic map of the powdery mildew fungus, blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley, is presented. the linkage analysis was conducted on 81 segregating haploid progeny isolates from a cross between 2 isolates differing in seven avirulence genes. a total of 359 loci were mapped, comprising 182 amplified fragment length polymorphism markers, 168 restriction fragment length polymorphism markers including 42 ltr-retrotransposon loci and 99 expressed sequence tags ( ...200211929213
the dna damage response in filamentous fungi.the mechanisms used by fungal cells to repair dna damage have been subjects of intensive investigation for almost 50 years. as a result, the model yeasts schizosaccharomyces pombe and saccharomyces cerevisiae have led the way in yielding critical insights into the nature of the dna damage response. at the same time, largely through the efforts of etta kafer, hirokazu inoue, and colleagues, a substantial collection of aspergillus nidulans and neurospora crassa dna repair mutants has been identifi ...200211929209
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
evolution of the fot1 transposons in the genus fusarium: discontinuous distribution and epigenetic inactivation.to understand the evolution of fot1, a member of the pogo family widely dispersed in ascomycetes, we have performed a phylogenetic survey across the genus fusarium divided into six sections. the taxonomic distribution of fot1 is not homogeneous but patchy; it is prevalent in the fusarium oxysporum complex, absent in closely related sections, and found in five species from the most distant section martiella. multiple copies of fot1 were sequenced from each strain in which the element occurs. in t ...200211919292
the fle1 gene encoding a c2h2 zinc finger protein co-ordinates male and female sexual differentiation in podospora anserina.the flexuosa (fle1-1) mutant, isolated in podospora anserina, displays vegetative defects and two antagonistic sexual phenotypes: it produces several 1000-fold fewer microconidia (male gametes) than the wild-type strain and, conversely, more abundant protoperithecia (female organs). cloning and sequencing of the fle1 gene and of cdna identified an open reading frame encoding a 382-amino-acid polypeptide with two c2h2 zinc finger motifs. the predicted fle1 protein shares 46% identity with the flb ...200211918811
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
identification and characterization of the genes encoding the core histones and histone variants of neurospora crassa.we have identified and characterized the complete complement of genes encoding the core histones of neurospora crassa. in addition to the previously identified pair of genes that encode histones h3 and h4 (hh3 and hh4-1), we identified a second histone h4 gene (hh4-2), a divergently transcribed pair of genes that encode h2a and h2b (hh2a and hh2b), a homolog of the f/z family of h2a variants (hh2az), a homolog of the h3 variant cse4 from saccharomyces cerevisiae (hh3v), and a highly diverged h4 ...200211901114
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
mutants of the carotene cyclase domain of al-2 from neurospora crassa.phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. the cyclase function has been demonstrated for the products of the genes crtyb from the basidiomycete xanthophyllomyces dendrorhous, and for carra and carrp from the zygomycetes phycomyces blakesleeanus and mucor circinelloides, respectively. these three genes are highly similar to al-2 from neurospora crassa. ta ...200211862485
trna-like recognition of group i introns by a tyrosyl-trna synthetase.the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) functions in splicing group i introns by promoting the formation of the catalytically active rna structure. previous work suggested that cyt-18 recognizes a conserved trna-like structure of the group i intron catalytic core. here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and c-terminal domains of cyt-18 interact with the expected group i intron cognates of the aminoacyl-acceptor st ...200211854463
ds-like restless deletion derivatives occur in tolypocladium inflatum and two foreign hosts, neurospora crassa and penicillium chrysogenum.single copies of the transposon restless from tolypocladium inflatum were introduced into neurospora crassa and penicillium chrysogenum. excision of restless from its donor site was investigated in n. crassa and in p. chrysogenum using direct selective conditions. in n. crassa, forward selection was also analyzed. deleted restless elements were frequently obtained in addition to the expected complete removal of restless from its donor site. similar deleted elements were also identified in t. inf ...200211848679
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
identification and characterization of novel cytochrome p450 genes from the white-rot basidiomycete, coriolus versicolor.using a reverse-transcription polymerase chain reaction (rt-pcr) technique, cytochrome p450 genes were cloned from the lignin-degrading basidiomycete, coriolus versicolor. one possible p450 gene was identified, which consisted of 1,672 nucleotides and a poly(a) tail and encoded a deduced protein containing 449 amino acids. the deduced amino acid sequence revealed the presence of the p450 heme-binding motif, strongly suggesting that this protein belongs to the p450 superfamily, then designated cy ...200211831480
the oxa1 protein forms a homooligomeric complex and is an essential part of the mitochondrial export translocase in neurospora crassa.the oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. oxa1 was identified in yeast as a crucial component of the protein export machinery known as the oxa translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. we have identified the neurospora crassa oxa1 protein which shows a sequence identity of 22% to the yeast homologue. despite the low level of identity, the function of the homologues is conserved as th ...200211823466
osmoregulation and fungicide resistance: the neurospora crassa os-2 gene encodes a hog1 mitogen-activated protein kinase homologue.neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% nacl. we found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. to understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (map) kinase homologous to hog1 and can complement the osmosensitive ...200211823187
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
targeting vectors for gene diversification by meiotic recombination in neurospora crassa.we have constructed a pair of vectors, pdv2 and pdv3, that enable targeted insertion of exogenous dna into linkage group i of neurospora crassa at the his-3 locus. transplaced sequences are inserted between his-3 and the cog(l) recombination hot spot and include his-3 mutations that allow meiotic recombination initiated by cog(l) to be monitored. selection of correctly placed transforming dna is based on complementation between different his-3 alleles borne by the plasmids and transformation hos ...200211798282
protein import into mitochondria.most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol. protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria. the outer membrane contains one general translocase, the tom complex, whereas three distinct translocases are located in the inner membrane, whic ...200111798021
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
analysis of genes expressed during rice-magnaporthe grisea interactions.expressed sequence tag (est) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. a total of 511 clones was sequenced from a cdna library constructed from rice leaves (oryza sativa cv. nipponbare) infected with m. grisea strain 70-15 to generate 296 nonredundant ests. the sequences of 293 clones (57.3%) significantly matched national c ...200111763134
the role of g protein alpha subunits in the infection process of the gray mold fungus botrytis cinerea.to identify signal transduction pathways of the gray mold fungus botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (pcr)-based approach to isolate two genes (bcg1 and bcg2) encoding alpha subunits of heterotrimeric gtp-binding proteins. both genes have homologues in other fungi: bcg1 is a member of the g alpha(i) class, whereas bcg2 has similarities to the magc gene of magnaporthe grisea and the gna-2 gene of neurospora crassa. revers ...200111763127
mips: a database for genomes and protein sequences.the munich information center for protein sequences (mips-gsf, neuherberg, germany) continues to provide genome-related information in a systematic way. mips supports both national and european sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequence ...200211752246
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
amplified uvra protein can ameliorate the ultraviolet sensitivity of an escherichia coli reca mutant.when a reca strain of escherichia coli was transformed with the multicopy plasmid psf11 carrying the uvra gene of e. coli, its extreme ultraviolet (uv) sensitivity was decreased. the sensitivity of the lexa1 (ind(-)) strain to uv was also decreased by psf11. the reca cells expressing neurospora crassa uv damage endonuclease (uvde), encoding uv-endonuclease, show uv resistance. on the other hand, only partial amelioration of uv sensitivity of the reca strain was observed in the presence of the pl ...200111738941
identification of transcriptionally expressed pheromone receptor genes in filamentous ascomycetes.detection of pheromone genes in filamentous ascomycetes implicated the presence of pheromone receptor genes. similar to yeasts and basidiomycetes, these might be involved in a g-protein triggered signal transduction pathway during mating. we have identified two pheromone receptor genes, named pre1 and pre2, in the genome of the heterothallic filamentous ascomycete neurospora crassa and the closely related homothallic sordaria macrospora. the deduced pre1 gene product is a putative seven-transmem ...200111738813
identification of genes in the bw/be regulatory cascade in ustilago maydis.in the phytopathogenic fungus ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bw and be homeodomain proteins. to identify genes in the regulatory cascade triggered by the bw/be heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. at different time-points after induction, genes that are switched on or off were identified throug ...200111737646
homologous expression and characterization of cel61a (eg iv) of trichoderma reesei.there are currently four proteins in family 61 of the glycoside hydrolases, from trichoderma reesei, agaricus bisporus, cryptococcus neoformans and neurospora crassa. the enzymatic activity of these proteins has not been studied thoroughly. we report here the homologous expression and purification of t. reesei cel61a [previously named endoglucanase (eg) iv]. the enzyme was expressed in high amounts with a histidine tag on the c-terminus and purified by metal affinity chromatography. this is the ...200111737205
molecular and kinetic study of catalase-1, a durable large catalase of neurospora crassa.catalase-1 (cat-1), one of the two monofunctional catalases of neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. like most monofunctional catalases, cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a ph range of 4-12, and inactivated by 3-amino-1,2,4-triazole. unlike most monofunctional catalases, cat-1 consists of 88 kda monomers t ...200111728803
partial amino acid sequences of peptidyl-prolyl isomerases offusarium sporotrichioides.peptidyl-proprylyl cis-trans isomerase (ppiase) activity was observed from crude extract of fusarium sporotrichioides. proteins from this fungi were separated by two-dimensional polyacrylamide gel electrophoresis and more than one thousand protein spots were separated. two cytosolic ppiases were found by the n-terminal sequencing from the two separated spots. the n-terminal 41 residues of the major protein spot showed high sequence identity (63.4%) with ppiase from neurospora crassa. this protei ...199511725072
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
conversion of pipecolic acid into lysine in penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase.pipecolic acid is a component of several secondary metabolites in plants and fungi. this compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. in penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. we have isolated two p. chrysogenum mutants, named 7.2 and 10.25, that are una ...200111717275
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
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