blue light adaptation and desensitization of light signal transduction in neurospora crassa.the ascomycete neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. here, we present an investigation of this photoadaptation phenomenon. we demonstrated previously that two proteins of the neurospora blue light signal transduction chain, wc1 and wc2, are subject to light-dependent phosphorylation. wc1 was phosphorylated in parallel with the transient increase in transcript levels of light-regulated ...200111251826
the hydrophobin eas is largely unstructured in solution and functions by forming amyloid-like structures.fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. these monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. the molecular details underlying the formation of these monolayers, however, have been elusive. we have studied eas, the hydrophobin from ...200111250193
analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of neurospora crassa reveals novel clock-controlled an effort to determine genes that are expressed in mycelial cultures of neurospora crassa over the course of the circadian day, we have sequenced 13,000 cdna clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. contig analysis allowed the identification of 445 unique expressed sequence tags (ests) and 986 ests present in multiple cdna clones. for approximately 50% of the sequences (710 of 1431), significant matches to sequence ...200111238394
ods2: a multiplatform software application for creating integrated physical and genetic maps.a contig map is a physical map that shows the native order of a library of overlapping genomic clones. one common method for creating such maps involves using hybridization to detect clone overlaps. false- positive and false-negative hybridization errors, the presence of chimeric clones, and gaps in library coverage lead to ambiguity and error in the clone order. genomes with good genetic maps, such as neurospora crassa, provide a means for reducing ambiguities and errors when constructing conti ...200111238393
hybridization-based mapping of neurospora crassa linkage groups ii and part of the german neurospora crassa genome project, physical clone maps of linkage groups ii and v of n. crassa were generated by hybridization-based mapping. to this end, two different types of clone library were used: (1) a bacterial artificial clone library of 15-fold genome coverage and an average insert size of 69 kb, and (2) three cosmid libraries--each cloned in a different vector--with 17-fold coverage and 34 kb average insert size. for analysis, the libraries were arrayed on filters ...200111238391
the goodwin model: simulating the effect of light pulses on the circadian sporulation rhythm of neurospora crassa.the goodwin oscillator is a minimal model that describes the oscillatory negative feedback regulation of a translated protein which inhibits its own transcription. now, over 30 years later this scheme provides a basic description of the central components in the circadian oscillators of neurospora, drosophila, and mammals. we showed previously that neurospora's resetting behavior by pulses of temperature, cycloheximide or heat shock can be simulated by this model, in which degradation processes ...200111237568
a conserved mechanism for post-transcriptional gene silencing?proteins with homology to rna-directed rna polymerases function in post-transcriptional gene silencing: in quelling in the fungus neurospora crassa, rnai in the nematode caenorhabditis elegans, and co-suppression in the mustard plant arabidopsis thaliana. these findings are consistent with a conserved mechanism operating in these diverse species.200011178243
repeat-induced point mutation (rip) in crosses with wild-isolated strains of neurospora crassa: evidence for dominant reduction of rip.seventy-one wild-isolated strains of neurospora crassa were examined for their ability to support repeat-induced point mutation (rip) in the erg-3 locus. rip was exceptionally inefficient but detectable in crosses with the strain fgsc 430 from adiopodoume, ivory coast. we could find no consistent differences in ascospore yields when wild isolates identified as "low-rip" or "high-rip" strains were crossed with strains bearing the segmental duplication dp(iiir > [i; ii])ar17. this suggested that r ...200011170738
identification of specificity determinants and generation of alleles with novel specificity at the het-c heterokaryon incompatibility locus of neurospora crassa.the capacity for nonself recognition is a ubiquitous and essential aspect of biology. in filamentous fungi, nonself recognition during vegetative growth is believed to be mediated by genetic differences at heterokaryon incompatibility (het) loci. filamentous fungi are capable of undergoing hyphal fusion to form mycelial networks and with other individuals to form vegetative heterokaryons, in which genetically distinct nuclei occupy a common cytoplasm. in neurospora crassa, 11 het loci have been ...200111158292
circadian regulation of the light input pathway in neurospora crassa.frequency (frq) is a critical element of the circadian system of neurospora. the white collar genes are important both for light reception and circadian function. we show that the responsiveness of the light input pathway is circadianly regulated. this circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. frq interacts genetically and physically with white collar-1, and physically with white collar-2. these findings begi ...200111157738
a neurospora crassa arp1 mutation affecting cytoplasmic dynein and dynactin localization.of the actin-related proteins, arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. the role of arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. usin ...200011129047
ph homeostasis of the circadian sporulation rhythm in clock mutants of neurospora crassa.the influence of environmental (extracellular) ph on the sporulation rhythm in neurospora crassa was investigated for wild-type (frq+) and the mutants chr, frq1, frq7, and frq8. in all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular ph in the range 4-9. on the other hand, for the same ph range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq1. a loss of ph homeostasis of the period, however, was observ ...200011128290
heat shock protein 80 of neurospora crassa: sequence analysis of the gene and expression during the asexual phase.heat shock protein 80 (hsp80) of neurospora crassa, a member of the stress-90 protein family, is a cytosolic molecular chaperone that interacts directly with hsp70 to form a hetero-oligomeric complex. the complete nucleotide sequence of the gene encoding this protein, along with the 5'- and 3'-flanking dna, is reported. the coding sequence is interrupted by two introns, 61 and 30 nucleotides, respectively, in length. the deduced amino acid sequence corresponds to a 695-residue polypeptide with a ...200011109485
in vivo chitin-cadmium complexation in cell wall of neurospora crassa.fungal cell wall, mainly composed of chitin, an n-acetylglucosamine polymer, is known to participate in heavy metal detoxification. in the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of neurospora crassa. based on the results of physical techniques, such as solid-state 13c-nmr, x-ray diffraction, ir and molecular modeling, a structure was proposed for the chitin-cadmium complex. the ring and c-3 hydroxyl oxygens ...200011099854
the external calcium-dependent nadph dehydrogenase from neurospora crassa mitochondria.we have inactivated the nuclear gene coding for a putative nad(p)h dehydrogenase from the inner membrane of neurospora crassa mitochondria by repeat-induced point mutations. the respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using nadh or nadph as substrates under different assay conditions. the results showed that the mutant lacks an external calcium-dependent nadph dehydrogenase. the observation of nadh and nadph oxidation by intact mitochondria from the nde ...200111073955
molecular and functional analyses of incompatibility genes at het-6 in a population of neurospora crassa.two closely linked genes, un-24 and het-6, associated with the het-6 heterokaryon incompatibility functional haplotype were examined in 40 neurospora crassa strains from a louisiana sugarcane field. partial diploid analyses were used to determine that half of the strains were functionally oak ridge (or) and half were non-or and indistinguishable from the standard panama (pa) form. pcr-based markers were developed to identify polymorphisms within both un-24 and het-6. two common forms of each gen ...200011035941
a novel type of fkbp in the secretory pathway of neurospora crassa.fkbps define a subfamily of peptidyl-prolyl cis/trans isomerases (pplases). pplases are known to play roles in cellular protein folding, protein interactions and signal transduction. here we describe ncfkbp22 from neurospora crassa, a novel type of fkbp. ncfkbp22 is synthesized as a precursor protein with a cleavable signal sequence. in addition to a typical fkbp domain in the amino-terminal part mature ncfkbp22 contains a novel second domain which is unique amongst all known fkbps. the amino ac ...200011034311
potassium distribution in the neurospora crassa hypha. 201611029033
respiratory chain complex i is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly.we have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex i from neurospora crassa, homologue of the mammalian tyky protein. analysis of the obtained mutant nuo21.3c revealed that complex i fails to assemble. the peripheral arm of the enzyme is disrupted while its membrane arm accumulates. furthermore, mutated 21.3c-kd proteins, in which selected cysteine residues were substituted with alanines or serines, were expresse ...200011014810
characterization of an unusual trna-like sequence found inserted in a neurospora retroplasmid.we characterized an unusual trna-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of neurospora intermedia strain varkud. we previously identified two forms of the trna-like sequence, one of 64 nt (trl-64)and the other of 78 nt (trl-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear trna intron. here, we show that trl-78 is encoded in varkud mitochondrial (mt)dna within a 7 kb sequence that is n ...200011001704
cellular distribution of cot1 kinase in neurospora crassa.the neurospora crassa cot-1 gene encodes a ser/thr protein kinase, which is involved in hyphal elongation. many vacuoles, abnormally shaped mitochondria, and nuclei, along with differences in the structure of the cell wall and hyphal septa, were observed in hyphae of the cot-1 mutant shortly after a shift to the restrictive temperature. immunolocalization experiments indicated that cot1 was associated with the cytoplasmic membrane; cot1 was also detected in the cytoplasm. the membrane-associated ...200010955908
a putative spectrin-containing membrane skeleton in hyphal tips of neurospora crassa.the apical plasma membrane (pm) is important in hyphal tip growth, where it may regulate tip extensibility via its association with an appropriate membrane skeleton (ms). by cell fractionation and immunocytochemistry we show that proteins with characteristics of actin, spectrin, and integrin are associated in a ms-like manner with the pm of neurospora crassa hyphae. the spectrin-like protein in particular is highly concentrated at the pm in the region of maximum apical expansion. this protein sh ...200010955906
non-coordinate regulation of 5s rrna genes and the gene encoding the 5s rrna-binding ribosomal protein homolog in neurospora eukaryotes, the levels of ribosomal proteins are coordinately regulated under varying nutritional conditions and at different developmental stages. little is known about how ribosomal protein levels are coupled to the levels of rrna. the formation of a ribonucleoprotein particle composed of 5s rrna and a ribosomal protein is an early step in ribosome assembly. to investigate how these two ribosomal components are regulated in neurospora crassa, we cloned the gene encoding the 5s rrna-binding ...200010954084
exploitation of a cellulose-binding domain from neurospora crassa.after eight decades as a purely research organism, neurospora crassa is becoming a production system for heterologous peptides. the present work exploits the cbh-1 gene, which encodes a class c cellobiohydrolase (ec and has, at its carboxy-terminus, a domain with homology to other fungal cellulose-binding domains. we describe the construction of two translational fusions of the putative cellulose-binding domain with a reporter gene, which is the catalytic domain of the gla-1 glucoamyla ...200010938423
cellular responses and repair of single-strand breaks introduced by uv damage endonuclease in mammalian cells.although single-strand breaks (ssbs) occur frequently, the cellular responses and repair of ssb are not well understood. to address this, we established mammalian cell lines expressing neurospora crassa uv damage endonuclease (uvde), which introduces a ssb with a 3'-oh immediately 5' to uv-induced cyclobutane pyrimidine dimers or 6-4 photoproducts and initiates an alternative excision repair process. xeroderma pigmentosum group a cells expressing uvde show uv resistance of almost the wild-type l ...200010924509
properties of a constitutive alkaline phosphatase from strain 74a of the mold neurospora crassa.a constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74a of the mold neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (pi) for 72 h at 30 degrees c. the molecular mass was 58 kda and 56 kda as determined by exclusion chromatography and sds-page, respectively. this monomeric enzyme shows an apparent optimum ph ranging from 9.5 to 10.5 a ...200010920432
senescent: a new neurospora crassa nuclear gene mutant derived from nature exhibits mitochondrial abnormalities and a "death" phenotype.fungi are capable of potentially unlimited growth. we resolved nuclear types from multinuclear mycelium of a phenotypically normal wild isolate of the fungus neurospora intermedia by plating its uninucleate microconidia and obtained a strain which, unlike the "parent" strain, exhibited clonal senescence in subcultures. the mutant gene, senescent, was introgressed into n. crassa and mapped four map units to the right of the his-1 locus on linkage group vr. senescent is the first nuclear gene muta ...200010882533
dynein and dynactin deficiencies affect the formation and function of the spitzenkörper and distort hyphal morphogenesis of neurospora crassa.the impact of mutations affecting microtubule-associated motor proteins on the morphology and cytology of hyphae of neurospora crassa was studied. two ropy mutants, ro-1 and ro-3, deficient in dynein and dynactin, respectively, were examined by video-enhanced phase-contrast microscopy and image analysis. in contrast to the regular, hyphoid morphology of wild-type hyphae, the hyphae of the ropy mutants exhibited a great variety of distorted, non-hyphoid morphologies. the ropy hyphae were slow-gro ...200010878138
use of an isocitrate lyase promoter-gfp fusion to monitor carbon metabolism of the plant pathogen tapesia yallundae during infection of wheat.abstract green fluorescent protein (gfp) has been used as a vital marker in a variety of species. here, we present the use of a gfp-promoter fusion to visualize carbon metabolism in a pathogenic fungus during growth on defined medium and during infection of plants. isocitrate lyase (icl), a key enzyme in carbon metabolism, is tightly regulated at the transcriptional level, with high levels of expression during 2-carbon growth and no expression during growth on glucose. a gfp-icl promoter fusion ...200020572972
short tpa-rich segments of the zeta-eta region induce dna methylation in neurospora crassa.the mechanisms that establish dna methylation in eukaryotes are poorly understood. in principle, methylation in a particular chromosomal region may reflect the presence of a "signal" that recruits methylation, the absence of a signal that prevents methylation, or both. experiments were carried out to address these possibilities for the 1.6 kb zeta-eta (zeta-eta) region, a relict of repeat-induced point mutation (rip) in the fungus neurospora crassa. the zeta-eta region directs its own de novo me ...200010873464
the cyanide-resistant alternative oxidases from the fungi pichia stipitis and neurospora crassa are monomeric and lack regulatory features of the plant enzyme.both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for atp synthesis. the dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. when this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids. a cys in the n-terminal section of the protein is responsible for both ...200010860541
sn-1,2-diacylglycerol levels in the fungus neurospora crassa display circadian rhythmicity.the fungus neurospora crassa is a model organism for investigating the biochemical mechanism of circadian (daily) rhythmicity. when a choline-requiring strain (chol-1) is depleted of choline, the period of the conidiation rhythm lengthens. we have found that the levels of sn-1,2-diacylglycerol (dag) increase in proportion to the increase in period. other clock mutations that change the period do not affect the levels of dag. membrane-permeant dags and inhibitors of dag kinase were found to furth ...200010859307
an rna-dependent rna polymerase gene in arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and rna interference in animals. here, we describe four genetic loci that are required for posttranscriptional gene silencing in arabidopsis. one of these, sde1, is a plant homolog of qde-1 in neurospora crassa that encodes an rna-dependent rna polymerase. the sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. we propose that the ro ...200010850496
senescence associated with the over-replication of a mitochondrial retroplasmid in neurospora crassa.mitochondrial dna rearrangements and deletions are a prevailing feature of filamentous fungal cultures that undergo senescence. in neurospora spp., strains containing the mauriceville and varkud mitochondrial retroplasmids routinely senesce at elevated temperatures, a process that is initiated by the integration of variant forms of the plasmids into the mitochondrial genome. here, we describe a strain that is phenotypically distinguishable from previously characterized senescent strains and show ...200010821177
comparative analysis of the mating-type loci from neurospora crassa and sordaria macrospora: identification of novel transcribed orfs.the mating-type locus controls mating and sexual development in filamentous ascomycetes. in the heterothallic ascomycete neurospora crassa, the genes that confer mating behavior comprise dissimilar dna sequences (idiomorphs) in the mat a and mat a mating partners. in the homothallic fungus sordaria macrospora, sequences corresponding to both idiomorphs are located contiguously in the mating-type locus, which contains one chimeric gene, smt a-3, that includes sequences which are similar to sequen ...200010778748
polyamine regulation of ornithine decarboxylase synthesis in neurospora crassa.ornithine decarboxylase (odc) of the fungus neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. in n. crassa, polyamines repress the synthesis and increase the degradation of odc protein. changes in the rate of odc synthesis correlate with similar changes in the abundance of spe-1 mrna. we identify two sequence elements, one in each of the 5' and 3' regions of the spe-1 gene of n. crassa, requi ...200010733579
characterization of an unusual trna-like sequence found inserted in a neurospora retroplasmid.we characterized an unusual trna-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of neurospora intermedia strain varkud. we previously identified two forms of the trna-like sequence, one of 64 nt (trl-64) and the other of 78 nt (trl-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear trna intron. here, we show that trl-78 is encoded in varkud mitochondrial (mt)dna within a 7 kb sequence that is ...200010710417
ego-1 is related to rna-directed rna polymerase and functions in germ-line development and rna interference in c. elegans.cell-fate determination requires that cells choose between alternative developmental pathways. for example, germ cells in the nematode worm caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. germ-line mitosis depends on a somatic signal that is mediated by a notch-type signaling pathway. the ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for o ...200010704412
nadh dehydrogenase in neurospora crassa contains myristic acid covalently linked to the nd5 subunit peptide.the mitochondrial, proton-pumping nadh:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex i catalyzes the first steps of mitochondrial electron transfer and proton translocation. radiolabel from [(3)h]myristic acid was incorporated by neurospora crassa into the mitochondrial-encoded, approximately 70 kda nd5 subunit of nadh dehydrogenase, as shown by immunoprecipitation. this myristate apparently was linked to ...200010699461
cryo-electron tomography of neurospora mitochondria.cryo-electron tomography was used to study the structural organization of whole frozen-hydrated mitochondria from neurospora crassa. unlike mitochondria from many other species and tissues, in this case the cristae form a three-dimensional network of interconnected lamellae. basically, the three-dimensional structure of ice-embedded mitochondria from this species is consistent with previous descriptions of mitochondria prepared by chemical fixation and resin embedding. nonetheless, ice-embedded ...200010675296
an osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in neurospora osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by repeat induced point mutation (rip) from a cross between a wild-type n. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group ii (lgii). the mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. dna transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. a ...200010660063
purification and characterization of flavokinase from neurospora crassa.the atp-dependent phosphorylation of riboflavin to fmn by flavokinase is the key step in flavin biosynthesis. flavokinase has been purified from a fungal source for the first time. the enzyme purified from a cell wall lacking mutant of neurospora crassa, slime, is a monomer of m(r) 35.5 kda with maximal activity at alkaline ph and high temperature (55 degrees c). the k(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nm for riboflavin and 210 nm for mgatp ...199910650711
the goodwin oscillator: on the importance of degradation reactions in the circadian clock.this article focuses on the goodwin oscillator and related minimal models, which describe negative feedback schemes that are of relevance for the circadian rhythms in neurospora, drosophila, and probably also in mammals. the temperature behavior of clock mutants in neurospora crassa and drosophila melanogaster are well described by the goodwin model, at least on a semi-quantitative level. a similar semi-quantitative description has been found for neurospora crassa phase response curves with resp ...199910643743
localization and light-dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in neurospora neurospora crassa only two white collar (wc) mutants, wc-1 and wc-2, have been described that seem to be insensitive to light. the pleiotropic phenotypes of these mutants suggest that they represent two central components of blue light signal transduction. the wc proteins have several characteristics of transcription factors consistent with an involvement in transcriptional control of light-regulated genes. here, we present a biochemical analysis of wc1 and wc2 polypeptides in n. crassa. usin ...200010632711
circadian rhythms in neurospora crassa: lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants.the conidiation rhythm in the fungus neurospora crassa is a model system for investigating the genetics of circadian clocks. null mutants at the frq (frequency) locus (frq(9) and frq(10)) make no functional frq gene products and are arrhythmic under standard conditions. the white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. all three genes are proposed to be central components of the circadian oscillator. we have been investigating two mutants ...200010618405
disruption of vma-1, the gene encoding the catalytic subunit of the vacuolar h(+)-atpase, causes severe morphological changes in neurospora using the process of repeat-induced point mutation (selker, e. u., and garrett, p. w. (1988) proc. natl. acad. sci. u. s. a. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit a of the v-atpase of neurospora crassa. two vma-1 mutant strains were characterized. one was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. the other had four point mutations, made a protein product, and produced viable spores. neither strain had ...200010617601
induced revertants of biochemical mutants of neurospora crassa: personal recollections. 199910610431
learning to "think like a geneticist" with the de serres ad-3 forward-mutation test in neurospora crassa. 199910610427
crystallization, structure and dynamics of the proton-translocating p-type atpase.large single three-dimensional crystals of the dodecylmaltoside complex of the neurospora crassa plasma membrane h(+)-atpase (h(+) p-atpase) can be grown in polyethylene-glycol-containing solutions optimized for moderate supersaturation of both the protein surfaces and detergent micellar region. large two-dimensional h(+) p-atpase crystals also grow on the surface of such mixtures and on carbon films located at such surfaces. electron crystallographic analysis of the two-dimensional crystals gro ...200010600683
cellular role of the v-atpase in neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit a.vacuolar atpases (v-atpases) are large complex enzymes that are structural and mechanistic relatives of f(1)f(o)-atpases. they hydrolyze atp and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. the proton gradients generated are used to drive the movement of various compounds across membranes. v-atpases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the p ...200010600678
recombinational landscape across a 650-kb contig on the right arm of linkage group v in neurospora crassa.we have established a 650-kb walk spanning the sp to ure-1 interval on neurospora crassa linkage group v. loci covered by the walk are, from proximal to distal, sp; rec-2; ure-2; dna polymerase delta; am; gul-1; ace-5; and ure-1. we have found extensive dna polymorphism in this region and used this to examine the recombinational landscape. crossovers are not evenly distributed over the region covered by the contig.199910591967
isolation and characterization of neurospora crassa nucleoside diphosphate kinase ndk-1.we have previously reported that phosphorylation of a 15-kda protein increased after blue-light irradiation in neurospora crassa. in this study, the 15-kda protein was purified using four columns; deae-cellulose, blue-sepharose, sp-sepharose and mono q. the 15-kda protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. a gene encoding n. crassa nucleoside diphosphate kinase, ndk-1, was is ...199910583364
the tom core complex: the general protein import pore of the outer membrane of mitochondria.translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane tom complex. we have isolated the tom core complex of neurospora crassa by removing the receptors tom70 and tom20 from the isolated tom holo complex by treatment with the detergent dodecyl maltoside. it consists of tom40, tom22, and the small tom components, tom6 and tom7. this core complex was also purified directly from mitochondria after solubilization with do ...199910579717
dna repair and specific-locus mutagenesis in neurospora crassa. 199910556617
involvement of protein kinase c in the response of neurospora crassa to blue a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. we tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. this approach has implicate ...199910517328
microscopic analysis of neurospora ropy mutants defective in nuclear distribution.movement and distribution of nuclei in fungi has been shown to be dependent on microtubules and the microtubule-associated motor cytoplasmic dynein. neurospora crassa mutants known as ropy are defective in nuclear distribution. we have shown that three of the ro genes, ro-1, ro-3, and ro-4, encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. three other ro genes, ro-7, ro-10, and ro-11, are required for the integrity or localization of cytoplasmic dynein or dy ...199910512672
characterization of a neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.we produced a photolyase-deficient mutant by repeat induced point mutation using the neurospora crassa photolyase gene cloned previously. this mutation identified a new gene, phr, which was mapped on the right arm of linkage group i by both rflp mapping and conventional mapping. to investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and ...199910512668
action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in neurospora crassa, dna sequence duplications are detected and altered efficiently during the sexual cycle by a process known as rip (repeat-induced point mutation). affected sequences are subjected to multiple gc-to-at mutations. to explore the pattern in which base changes are laid down by rip we examined two sets of strains. first, we examined the products of a presumptive spontaneous rip event at the mtr locus. results of sequencing suggested that a single rip event produces two distinct p ...199910511550
characterization of neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase.neurospora crassa cel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding beta-fatty acid synthase. these mutants are phenotypically less leaky than cel-1, which has a defective alpha-fatty acid synthase. the cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and synthesized less fatty acid de novo than cel-1. unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for f ...199910501937
inactivation of the neurospora crassa mitochondrial outer membrane protein tom70 by repeat-induced point mutation (rip) causes defects in mitochondrial protein import and morphology.mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the tom complex (translocase of the outer mitochondrial membrane). the largest component of this complex is tom70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. to investigat ...199910501936
a reductase/isomerase subunit of mitochondrial nadh:ubiquinone oxidoreductase (complex i) carries an nadph and is involved in the biogenesis of the complex.respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping nadh:ubiquinone oxidoreductase, or complex i. the bacterial complex i consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. one of these extra subunits with a molecular mass of 40 kda belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. we deleted this subun ...199910497022
effects of disrupting the 21 kda subunit of complex i from neurospora crassa.we have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kda subunit of complex i from neurospora crassa. mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex i in approximately normal amounts. in addition, this mutant is able to assemble an almost intact enzyme. the electron transfer activities from nadh to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type s ...199910477266
bioelectrorheological model of the cell. 8. cellular deformation under prolonged and recurrent shear stress.the influence of a prolonged and recurrent shear stress created by a periodic electric field on the mechanical properties of neurospora crassa cells was investigated. conditions were found under which modifications of cellular structures responding to stress become irreversible, and plastic flow of the viscoelastic structural elements is observed. the symmetry of the response of the cell under stress application and relaxation was lost, when compared to the reference conditions. to interpret the ...200810474657
the antibiotic viomycin as a model peptide for the origin of the co-evolution of rna and proteins.viomycin is an rna-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group i intron self-splicing. this antibiotic enhances the activity of the ribozyme derived from the neurospora crassa vs rna, and at sub-inhibitory concentrations it induces the formation of group i intron oligomers. here, we address the question whether viomycin exerts specificity in the promotion of rna-rna interactions. in an in vitro selection experiment we tested the ability of viomycin to specif ...199910472628
gene silencing: rna makes rna makes no protein.a mutation that disrupts post-transcriptional gene silencing in neurospora crassa has been found to affect the homologue of a plant-encoded rna-dependent rna polymerase. this enzyme may produce a specificity determinant of gene silencing and mediate an epigenetic conversion at the rna level.199910469584
kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in neurospora.kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. they have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions.201610469561
functional anatomy of the kinesin molecule in vivo.we have developed an assay that allows the functional efficiency of mutant kinesins to be probed in vivo. we show here that the growth rate of the filamentous fungus neurospora crassa can be used as a sensitive reporter for the ability of mutant kinesins to suppress the phenotype of the kinesin null mutant of neurospora. truncation mutants, internal deletion mutants and chimeras, in which homologous domains were exchanged between different fungal kinesins, were generated and transformed into the ...199910449406
a mutation within the catalytic domain of cot1 kinase confers changes in the presence of two cot1 isoforms and in ser/thr protein kinase and phosphatase activities in neurospora crassa.neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of n. crassa colonial temperature-sensitive (cot) mutants and encodes a ser/thr protein kinase. we have mapped the cot-1 mutation to a single base change resulting in a his to arg substitution at amino acid 351, which resides within the catalytic domain. antibodies raised against cot1 detected and immunoprecipitated a predominant 73-kda polypeptide in n. crassa extracts, whose abundance was constant under all growth ...200910441452
nucleotide binding and hydrolysis properties of neurospora crassa cytosolic molecular chaperones, hsp70 and hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.formation of a hetero-oligomeric complex between hsp70 and hsp80 of neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (elisa). the present study documents the effect of nucleotides on the subunit structure of hsp70 and hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (dma), and dimethyl suberimidate (dms). the inter-protomer crosslinking of hsp80 with dma and dms was suppressed by atp and to a ...199910438143
the drosophila melanogaster gene for the nadh:ubiquinone oxidoreductase acyl carrier protein: developmental expression analysis and evidence for alternatively spliced forms.we have isolated the drosophila melanogaster gene encoding the mitochondrial acyl carrier protein (mtacp), a subunit of nadh:ubiquinone oxidoreductase involved in de novo fatty acid synthesis in the mitochondrion. this gene expresses two distinct mature transcripts by alternative splicing, which encode mature polypeptides of 86 (mtacp1a) and 88 (mtacp1b) amino acids, respectively. drosophila mtacp1 is 72% identical to mammalian mtacp, 47% identical to arabidopsis thaliana mtacp, and 46% identica ...199910394906
the nop-1 gene of neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. previously, genes encoding opsins had been identified in animals and the archaea but not in fungi or other eukaryotic microorganisms. here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus neurospora crassa. the nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid ...199910393943
evidence for negative interference: clustering of crossovers close to the am locus in neurospora crassa among am response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, fincham proposed that recombination events at the neurospora am locus rarely have an associated crossover. fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am i ...199910388816
assignment of circadian function for the neurospora clock gene frequency.circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. it is difficult to assign molecular clock components to any one of these elements. experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator. mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the p ...199910376598
neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution.movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. we have isolated hundreds of neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. in this report, we describe the isolation and initial characterization o ...199910361308
the structure of the vacuolar atpase in neurospora crassa.the filamentous fungus neurospora crassa contains many small vacuoles. these organelles contain high concentrations of polyphosphates and basic amino acids, such as arginine and ornithine. because of their size and density, the vacuoles can be separated from other organelles in the cell. the atp-driven proton pump in the vacuolar membrane is a typical v-type atpase. we examined the size and structure of this enzyme using radiation inactivation and electron microscopy. the vacuolar atpase is a la ...199910340846
gene silencing in neurospora crassa requires a protein homologous to rna-dependent rna plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. this phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. transgene-induced gene silencing has been termed quelling in neurospora crassa and co-suppression in plants. quelling-defective (qde) mutants of n. crassa, in which transgene-induced gene silencing is ...199910335848
functional analysis of the two zinc fingers of sre, a gata-type factor that negatively regulates siderophore synthesis in neurospora crassa.multiple gata factors, zinc finger dna binding proteins that recognize consensus gata elements, exist in neurospora crassa. one of them, sre, is involved in controlling the iron metabolic pathway of n. crassa. in n. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. the siderophore synthesis pathway is negatively regulated by sre; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. in the research presented here, ...199910194352
glucose metabolism in neurospora is altered by heat shock and by disruption of hsp30.we compared the metabolism of [1-13c]glucose by wild type cells of neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. high temperature led to increased incorporation of 13c into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees c). heat shock strongly reduced formation of tricarboxylic a ...199910076052
microconidia of neurospora crassa.neurospora crassa produces two types of vegetative spores-relatively small numbers of uninucleate microconidia and very large numbers of multinucleate macroconidia (blastoconidia and arthroconidia). the microconidia can function either as spermatia (male gametes) or as asexual reproductive structures or both. in nature they probably function exclusively in fertilization of protoperithecia. the environmental conditions favoring their formation and the pattern of their development are quite distin ...199910072316
the goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of neurospora crassa.the goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of neurospora and drosophila. an essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. when protein synthesis in n. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (chx) the period of the circadian sporulation rhythmicity ...199910036201
the b regulatory subunit of protein phosphatase 2a is required for completion of macroconidiation and other developmental processes in neurospora crassa.rgb-1, encoding the tentative b regulatory subunit of the type 2a ser/thr phosphatase in neurospora crassa, was isolated from cdna and genomic libraries. based on analysis of cdna and genomic clones, rgb-1 is 3387 nucleotides in length, contains seven putative introns and encodes a 461-amino-acid polypeptide. intron i, which is 5' to the presumed translation initiation codon, contains a uorf encoding 34 amino acids. intron vi undergoes alternative splicing. inactivation of rgb-1 by the repeat-in ...19999987122
molecular characterization of tol, a mediator of mating-type-associated vegetative incompatibility in neurospora crassa.the mating-type locus in the haploid filamentous fungus, neurospora crassa, controls mating and sexual development. the fusion of reproductive structures of opposite mating type, a and a, is required to initiate sexual reproduction. however, the fusion of hyphae of opposite mating type during vegetative growth results in growth inhibition and cell death, a process that is mediated by the tol locus. mutations in tol are recessive and suppress mating-type-associated heterokaryon incompatibility. i ...19999927450
search for novel redox groups in mitochondrial nadh:ubiquinone oxidoreductase (complex i) by diode array uv/vis spectroscopy.the proton-translocating nadh:ubiquinone oxidoreductase of mitochondria (complex i) is a large l-shaped multisubunit complex. the peripheral matrix arm contains one fmn and a number of iron-sulfur (fes) clusters and is involved in nadh oxidation and electron transfer to the membrane intrinsic arm. there, following a yet unknown mechanism, the redox-driven proton translocation and the ubiquinone reduction take place. redox groups that would be able to link electron transfer with proton translocat ...19989914816
folylpolyglutamate synthesis in neurospora crassa: primary structure of the folylpolyglutamate synthetase gene and elucidation of the met-6 neurospora crassa, the met-6+ gene encodes folylpoly-gamma-glutamate synthetase (fpgs) which catalyzes the formation of polyglutamate forms of folate. methionine auxotrophy of the neurospora crassa met-6 mutant is related to a lesion affecting this enzyme. functional complementation of the mutant strain was achieved by introducing copies of the wild-type met-6+ gene into mutant spheroplasts. the complementing sequences were found to be contained on a 3.5 kb ecori-bamhi restriction fragment. t ...19989887523
radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.a few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. the enzyme isoorotate decarboxylase (idcase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. we describe in this communication two assays for idcase and the ...19999887216
mutational activation of a galphai causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in neurospora crassa.heterotrimeric g proteins, consisting of alpha, beta, and gamma subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. we previously reported that the filamentous fungus neurospora crassa possesses a galpha protein, gna-1, that is a member of the galphai superfamily. deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. in addition, deltagna-1 strains have low ...19999872952
spermidine determines the sensitivity to the calmodulin antagonist, chlorpromazine, for the circadian conidiation rhythm but not for the mycelial growth in neurospora crassa.the gene that suppresses the phenotype of the cpz-2 mutation, which results in changing the sensitivity to chlorpromazine in relation to mycelial growth and circadian rhythms, was cloned in neurospora crassa. this gene is not the cpz-2 gene itself but rather is identical to the spe-3 gene that encodes spermidine synthase in neurospora. the intracellular content of spermidine was lowered in the cpz-2 strain compared to that of the wild-type strain. by integration of the spe-3 gene or by the addit ...19989850006
differential stress gene expression during the development of neurospora crassa and other fungi.stress genes are differentially expressed during the development of neurospora crassa and other fungi. large amounts of constitutive heat shock protein 70 (hsc70) are found in dormant conidia of n. crassa, whereas little mrna of the related glucose-regulated protein (grp78) is detected. it is, however, not generally clear whether heat shock protein or mrna is preferentially stored in dormant spores. germinating spores of n. crassa increase the level of grp mrna. during this developmental stage, ...19989835024
the evolutionarily conserved eukaryotic arginine attenuator peptide regulates the movement of ribosomes that have translated it.translation of the upstream open reading frame (uorf) in the 5' leader segment of the neurospora crassa arg-2 mrna causes reduced initiation at a downstream start codon when arginine is plentiful. previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous n. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uorf termination codon. this stalling apparently regulates translation by pr ...19989819438
the mating type locus of neurospora crassa: identification of an adjacent gene and characterization of transcripts surrounding the idiomorphs.complexity in and around the a and a mating type idiomorphs in neurospora crassa was examined. six sets of transcripts surrounding the idiomorphs were identified by northern analysis. several different patterns of regulation were observed. the two pairs of transcripts closest to the centromere-proximal idiomorph flanks (variable regions) exhibited mating type-specific size differences. dna sequence was obtained for the region surrounding the four transcripts which showed mating type-specific siz ...19989819054
temperature adaptation of house keeping and heat shock gene expression in neurospora crassa.adaptation of house keeping and heat shock gene expression was determined in neurospora crassa during continuous exposure to different temperatures. steady-state values of total protein synthesis differed little after incubation for 24 h at temperatures between 15 and 42 degreesc. adaptation kinetics at 42 degreesc showed an initial, transient inhibition of total protein synthesis. similar kinetics were observed with actin synthesis and tubulin mrna. a priming 1-h heat shock of 42 degreesc 2 h p ...19989806804
carbon catabolite repression of gene expression and conidiation in neurospora crassa. 19989806802
promoter analysis of the acetate-inducible isocitrate lyase gene (acu-3) from neurospora crassa.analysis of the promoter region of the acetate-induced isocitrate lyase gene (acu-3) of neurospora crassa was undertaken. a series of deletions in the 5' non-transcribed region were constructed and the effects of these mutations on the enzyme levels following growth on sucrose and transfer to acetate were measured. sequences within the region -603 to -271 relative to the transcription start site appear essential for transcription. the region -950 to -1278 is required for sucrose repression, whic ...19989804981
multiple forms of arginase are differentially expressed from a single locus in neurospora crassa.the neurospora crassa catabolic enzyme, arginase (l-arginine amidinohydrolase, ec, exists in multiple forms. multiple forms of arginase are found in many vertebrates, but this is the only reported example in a microbial organism. the two major forms are structurally similar with subunit sizes of 36 and 41 kda, respectively. the larger form is produced by mycelia growing in arginine-supplemented medium. both forms are localized in the cytosol. the structural gene for arginase, aga, has b ...19989792692
protein phosphatase 2a is involved in hyphal growth of neurospora crassa.cantharidin and calyculin a, natural toxins that are inhibitors of protein phosphatases 1 and 2a (ppi and pp2a, respectively). inhibit neurospora crassa hyphal growth. when n. crassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. in addition, both drugs induced an increase in hyphal branching. cantharidin inhibited n. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34 degrees c than at 25 degrees c. i ...19989790584
evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. as filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. however, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. on ...19989770498
a cell-free translation-translocation system reconstituted with subcellular fractions from the wall-less variant fz;sg;os-1v of neurospora crassa.a translation-translocation system reconstituted with subcellular fractions from the wall-less variant fz;sg;os-1v of neurospora crassa reproduces in vitro translocation and processing of a secretory protein. the translation extract was isolated from the wall-less variant by gently lysing cells by a freeze-thaw procedure. this method yielded more extract then the method developed previously (r. addison, j. biol. chem. 262: 17031, 1987) as well as reducing microsomal contamination. the microsomal ...19989756715
mutation of the gene for the second-largest subunit of rna polymerase i prolongs the period length of the circadian conidiation rhythm in neurospora crassa.the period length of the circadian conidiation rhythm was examined in a mutant strain of neurospora crassa, un-18, that is temperature sensitive for mycelial growth. the un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. below 22 degrees c, the un-18 mutation did not affect the period length, but at temperatures between 22 degrees c and 32 degrees c, the period length of the un-18 mutant was approximately 2 ...19989749669
what determines growth direction in fungal hyphae?we used high-resolution video microscopy and image analysis to map the trajectory of the spitzenkörper in growing hyphae of neurospora crassa and to correlate it with growth directionality. the spitzenkörper followed a tortuous trajectory produced by a dominant forward motion accompanied by frequent, transverse oscillations. in hyphae with a fixed growth direction, the regression line of the spitzenkörper trajectory corresponded to the longitudinal axis of the hypha. a permanent change in growth ...20079742196
mutations in the dim-1 gene of neurospora crassa reduce the level of dna methylation.mutants that show reduced dna methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azac) promotes the formation of potentially lethal dna-methyltransferase adducts; (ii) reduction in dna methyltransferase will decrease the sensitivity of cells to 5-azac; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azac-induced dna damage. of fifteen potential repair mutants screen ...19989738881
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