gtp is required for the integration of a fragment of the neurospora crassa h(+)-atpase into homologous microsomal vesicles.the integration of a fragment of the neurospora crassa plasma membrane h(+)-atpase was examined to determine if insertion of the fragment into homologous microsomal vesicles is obligatorily dependent on a nucleoside triphosphate. rna transcripts that encoded the amino terminal 344 amino acids of the neurospora crassa plasma membrane h(+)-atpase(pma(344)+) were translated in a n. crassa in vitro system. the pma(344)+ integrated post-translationally into homologous microsomal vesicles independent ...19911711898
the cdna sequence and expression of an ubiquitin-tail gene fusion in neurospora crassa.the genome of neurospora crassa contains at least one natural fusion gene encoding a single ubiquitin (ubi) unit with a 78-amino acid c-terminal extension. the predominantly basic tail sequence corresponds to a highly conserved ribosomal protein identified in other organisms. the 0.7-kb ubi fusion transcript is mainly expressed in germinating conidia and other stages of active cell replication. under starvation conditions attained by nutrient depletion, or after polyamine depletion, the ubi fusi ...19911650731
nit-3, the structural gene of nitrate reductase in neurospora crassa: nucleotide sequence and regulation of mrna synthesis and turnover.the nit-3 gene of the filamentous fungus neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. the nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kda. comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant ...19911829499
effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in neurospora crassa.neurospora crassa conidiospore germlings exposed to a heat shock (30-45 c) rapidly accumulated trehalose and degraded glycogen, even in the presence of cycloheximide. this phenomenon was also rapidly reversible upon return of the cells at 30 degrees c. trehalose accumulation at 45 degrees c demanded an exogenous source of carbon and either glucose or glycerol fulfilled such requirement. experiments with the cyclic amp-deficient cr-1 mutant suggested that the effects of temperature shifts on treh ...19911645296
isolation of uridine auxotrophs from trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes.uridine auxotrophs of the filamentous fungus trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. the homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. transformation efficiency with these homologous systems is very high (greater tha ...19911913875
regulation of laccase synthesis in induced neurospora crassa cultures.rapidly growing cultures of n. crassa do not produce laccase. exposure of this fungus to different inducing agents leads to a de novo biosynthesis of extracellular laccase in vegetative cultures. in this study the induction of laccase after addition of cycloheximide and d-phenylalanine is reported. de novo synthesis of laccase mrna was followed over 96 h after induction. a fast appearance of the message, as well as its presence over a rather long period, indicates a regulation on a transcription ...19911833078
determination of electric parameters of cell membranes by a dielectrophoresis method.marszalek, p., j. j. zielinsky, and m. fikus (1989. bioelectrochem. bioenerg. 22:289-298) have described a novel design for measuring the complete dielectrophoretic spectrum of a single cell. from the analysis of the dielectrophoretic spectrum, the membrane conductivity, sigma membr, and the membrane dielectric permittivity, epsilon membr, of the cell may be determined according to the theory of dielectrophoresis described by sauer, f. a. (1985. interactions between electromagnetic field and cel ...19911831052
photostimulation of conidiation in mutants of neurospora crassa.various mutants of neurospora crassa were screened for light-stimulated conidiation which is a blue light effect and, at least in strain albino-band, is mediated by the flavoprotein nitrate reductase (nr). nr- mutants showed practically no photoconidiation under standard conditions. however, in fusion products of nit-1 (diaphorase activity present, terminal activity missing) plus nit-3 (terminal activity present, diaphorase activity missing), nr activities and photoconidiation were partially res ...19911830899
loss of nad(p)-reducing power and glutathione disulfide excretion at the start of induction of aerial growth in neurospora crassa.when exponentially growing hyphae of neurospora crassa in aerated liquid cultures are filtered and the resulting mycelial mat is exposed to air, aerial hyphae develop and synchronous conidiation is obtained. the hyphae in direct contact with air adhere to each other within minutes and form aerial hyphae during the following 12 h; the hyphae which are not in direct contact with air do not adhere to each other and do not form aerial hyphae. previous data indicated that oxidative stress was generat ...19911827113
an inducible gene expression system for neurospora crassa.neurospora crassa acetyl coa synthetase is highly induced when the growing mycelium is transferred from sucrose- to acetate-based medium. the inducible promoter of this gene has been isolated and used to control the expression of glutamate dehydrogenase. transformants containing this expression cassette show gdh levels up to 25 times higher than the nontransformed host strain. this expression cassette will form the basis of a system of heterologous gene expression.19911369451
regulation of biosynthesis of l-amino acid oxidase by neurospora crassa.l-amino acid oxidase was purified from liquid cultures of neurospora crassa induced by l-phenylalanine, d-phenylalanine, atp and cycloheximide. although the four enzyme species isolated were found to differ in size and electrophoretic mobility, they were the product of a single gene as demonstrated by genomic southern analysis. northern analysis of total cellular rna showed a rapid increase of l-amino acid oxidase mrna in response to the different inducing agents studied. these data suggest that ...19911829425
metalloselenonein, the selenium analogue of metallothionein: synthesis and characterization of its complex with copper ions.we used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in neurospora crassa copper metallothionein. metalloselenonein binds 3 mol of cu(i) per mol. this adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. the circular dichroism spectrum of the copper-metalloselenonein complex shows a positive b ...19911826562
ca2+ calmodulin-dependent protein kinase activity in the ascomycetes neurospora crassa.deae-cellulose column chromatography of neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated pki, pkii, and pkiii. pkii activity is stimulated by ca2+ and neurospora or brain calmodulin. maximal stimulation was observed at 2 microm-free ca2+ and 1 microgram/ml of the modulator. the stimulatory effect of the ca(2+)-calmodulin complex was blocked by egta and by some calmodulin antagonists such as phenothiazine drugs or compound ...19911652680
regulation of molybdenum cofactor species in the green alga chlamydomonas reinhardtii.molybdenum cofactor (moco) of molybdoenzymes is constitutively produced in cells of the green alga chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. in soluble form, moco was found to be present in several forms: (i) as a low mr free species; (ii) bound to a moco-carrier protein of about 50 kda that could release moco to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of neurospora crassa, but not t ...19911826614
nucleotide and dna uptake by neurospora crassa: involvement of an uptake stimulating protein.the basal and dusf (dna-uptake-stimulating factor, described previously by schablik and szabó (1981) fems microbiol. lett. 10, 395-397) stimulated uptake of [3h]dna and radioactive nucleotides by neurospora crassa (fgsc 1118, slime) cell-wall-less strain was studied. the uptake of [3h]dna by the cells is a saturable and time-dependent process. the ph and temperature optimum for [3h]dna uptake are ph 7 and 27 degrees c, respectively. both basal and dusf-stimulated uptake of [3h]dna are inhibited ...19911826455
specificity of repeat-induced point mutation (rip) in neurospora: sensitivity of non-neurospora sequences, a natural diverged tandem duplication, and unique dna adjacent to a duplicated region.the process designated rip (repeat-induced point mutation) alters duplicated dna sequences in the sexual cycle of neurospora crassa. we tested whether non-neurospora sequences are susceptible to rip, explored the basis for the observed immunity to this process of a diverged tandem duplication that probably arose by a natural duplication followed by rip (the neurospora zeta-eta region), and investigated whether rip extends at all into unique sequences bordering a duplicated region. bacterial sequ ...19911827630
recurrence of repeat-induced point mutation (rip) in neurospora crassa.duplicate dna sequences in the genome of neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed rip (repeat-induced point mutation). rip occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. we investigated the extent to which sequences altered by rip remain susceptible to th ...19911827629
surface topography and molecular stoichiometry of the mitochondrial channel, vdac, in crystalline arrays.the mitochondrial outer membrane contains a protein, called vdac, that forms large aqueous pores. in neurospora crassa outer membranes, vdac forms two-dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (c. mannella, science 224, 165, 1984). fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each o ...19911725124
purification and characterization of glutamate decarboxylase from neurospora crassa conidia.l-glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of neurospora crassa, was purified to homogeneity from conidia. the purification procedure included ammonium sulfate fractionation and deae-sephadex and cellulose phosphate column chromatography. the final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. a single band coincident with enzyme activity was found on native 7.5% polyacrylamid ...19911825829
kinetic properties of de-repressible alkaline phosphatase (ec from neurospora alkaline phosphatase from n. crassa shows inhibition by pnp-p and a hyperbolic mixed-type inhibition by pi. both increasing concentrations of pi and decreases in assay ph abolished inhibition by the substrate. also, pi promoted polymerization of the enzyme molecule, whose effect may account for the inhibitory behaviour shown by the enzyme in the presence of low pi concentrations.199124424939
polyamine toxicity in neurospora crassa: protective role of the vacuole.we used mutant strains of neurospora crassa to define the discretionary capacity of this species for excess putrescine. the spe-3 mutant, which accumulates putrescine internally, and the puu-1 mutant, which accumulates toxic levels of putrescine from the medium, both sequestered large excesses of putrescine in vacuoles. concomitantly in puu-1, inorganic polyphosphate increased modestly and some of the monovalent cation of the vacuole was discharged. these two factors contribute to the increased ...19911832828
calcium modulation of polyamine transport is lost in a putrescine-sensitive mutant of neurospora crassa.putrescine transport in neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. we describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to ca2+. the wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)n,n'-tetraacetic acid. the mutant phenotype also ap ...19911832827
purification of nad glycohydrolase from neurospora crassa conidia by a polyclonal immunoadsorbent.nad glycohydrolase from neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. the procedure was easy, non-denaturating and suitable for repetitive use of the gel. the enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.19911828469
31p-nmr-studies on intracellular ph and metabolite concentrations in relation to the circadian rhythm, temperature and nutrition in neurospora crassa.perfused mycelia of neurospora crassa were analyzed in vivo with 31p-nmr. both the cytoplasmatic and the vacuolar ph and the concentrations of phosphate metabolites were followed up to 30 h under constant conditions. no circadian changes were detected. however, slight changes in the nutrition or oxygen supply induced distinct changes in the intracellular ph and in the concentrations of metabolites. an increase of temperature from 21 to 43 degrees c lowered the intracellular ph and the metabolite ...19911825790
nucleotide sequence, messenger rna stability, and dna recognition elements of cys-14, the structural gene for sulfate permease ii in neurospora crassa.the complete nucleotide sequence of the cys-14 gene which encodes sulfate permease ii, a member of the sulfur regulatory circuit, is presented. the cys-14 gene contains four introns with consensus splice site sequences and is transcribed from four closely spaced initiation sites located approximately 20 bp upstream of the atg initiation codon. the translated cys14 protein is composed of 781 amino acids with a molecular weight of 87,037 and contains 12 potential hydrophobic membrane-spanning doma ...19911825178
cdna and genomic dna sequence of the 21.3 kda subunit of nadh:ubiquinone reductase (complex i) from neurospora crassa.the primary structure of a nuclear-encoded subunit of the respiratory chain nadh:ubiquinone reductase (complex i) from neurospora crassa was determined by sequencing cdna, genomic dna and the n-terminus of the protein. the sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 da. the protein is synthesized without a cleavable presequence. it contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex i.19911825789
purification and properties of a casein kinase ii-like enzyme from neurospora crassa.a serine/threonine protein kinase was partially purified from neurospora crassa. its physical and catalytic properties were typical of casein kinase ii. in vitro, the kinase phosphorylated a calpain like protease from allomyces arbuscula with higher affinity than a mixture of caseins.19911827768
a new senescence-inducing mitochondrial linear plasmid in field-isolated neurospora crassa strains from india.several field-collected strains of neurospora crassa from the vicinity or aarey, bombay, india, are prone to precocious senescence and death. analysis of one strain, aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. the senescence-prone strains contain a 7-kb, linear, mitochondrial dna plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. the maranhar plasmid has inverted terminal rep ...19911648454
regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochrome c reductase by the membrane potential.the electron transfer reaction catalysed by mitochondrial ubiquinol:cytochrome c reductase is linked to the outwards translocation of protons with an h+ e- stoichiometry of 1 under non-membrane potential condition. the effect of the electrical membrane potential on the h+/e- stoichiometry was investigated. the enzyme was isolated from neurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of th ...19911847681
purification and properties of a membrane-bound insulin binding protein, a putative receptor, from neurospora crassa.the protein that is responsible for specific, high-affinity binding of insulin to the surface of neurospora crassa cells has been purified to homogeneity. the insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. insulin binding activity was purified from triton x-100 solubilized membranes in two steps: fplc on a monoq hr5/5 column; and affinity chromato ...19911824821
relationship between a subunit of nadh dehydrogenase (complex i) and a protein family including subunits of cytochrome reductase and processing protease of mitochondria.the primary structure of a 40 kda subunit of the respiratory chain nadh:ubiquinone reductase from neurospora crassa was determined by sequencing cdna, genomic dna and the n-terminus of the mature protein. the gene which is interrupted by 7 introns encodes a preprotein consisting of 375 amino acids with a 26 amino acid long presequence typical for a mitochondrial targeting signal. the sequence of the mature subunit shows conspicuous similarities to the recently [(1989) nature 339, 147-149] discov ...19911825202
nuclease-induced dna structural changes assessed by flow cytometry with the intercalating dye propidium iodide.a flow cytometric analysis of dna structural changes induced by cleavage with nucleases was performed on isolated hela nuclei by assessing changes in stainability with the dna-specific fluorochrome propidium iodide (pi). after mild digestion with dnase i, micrococcal nuclease, or with the single-strand-specific s1 and neurospora crassa nucleases, fluorescence intensity of nuclei stained with pi increased by about 15-30% above the value of undigested control samples. no significant modifications ...19912065557
sequence of the cloned pyr4 gene of trichoderma reesei and its use as a homologous selectable marker for transformation.we have cloned and sequenced the trichoderma reesei pyr4 gene encoding orotidine-5'-monophosphate decarboxylase. comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that t. reesei is closely related to cephalosporium acremonium and neurospora crassa. the cloned pyr4 gene has been used as a homologous selectable marker for transformation of t. reesei. the majority of transformants obtained with circular plasmid were mitotically unstable and contained ...19912036683
effect of activators and inhibitors on the activity of mitochondrial dna a concentration of 0.5 to 3 mmol/l, atp stimulates the activity of mitochondrial dna polymerase of neurospora crassa under the optimum reaction conditions; at higher concentrations, an inhibitory effect is observed. 4-chloromercuribenzoate (1 mmol/l), a thiol inhibitor, decreases the enzyme activity two-fold, while n-ethylmalcimide (2 mmol/l) has no effect. ethidium bromide (up to 10 mumol/l) and heparin (up to 0.4 micrograms/ml) reduce the activity by 60%. ddttp does not affect the dna polym ...19911841856
purification of neurospora crassa copper-metallothionein. 19911838130
amplitude model for the effects of mutations and temperature on period and phase resetting of the neurospora circadian oscillator.this paper analyzes published and unpublished data on phase resetting of the circadian oscillator in the fungus neurospora crassa and demonstrates a correlation between period and resetting behavior in several mutants with altered periods: as the period increases, the apparent sensitivity to resetting by light and by cycloheximide decreases. sensitivity to resetting by temperature pulses may also decrease. we suggest that these mutations affect the amplitude of the oscillator and that a change i ...19911837742
temperature compensation of the circadian rhythm in neurospora crassa: comments on the vicinal water hypothesis. 19911837741
genetics and molecular biology of neurospora crassa. 19911837201
the wilhelmine e. key 1989 invitational lecture. organization and regulation of the qa (quinic acid) genes in neurospora crassa and other neurospora crassa, five structural genes and two regulatory genes control the use of quinic acid as a carbon source. all seven genes are tightly linked to form the qa gene cluster. the entire cluster, which has been cloned and sequenced, occupies a continuous dna segment of 17.3 kb. three pairs of genes are divergently transcribed, including the two regulatory genes that are located at one end of the cluster and that encode an activator (qa-1f) and a repressor (qa-1s). three of the structural ...20131825499
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. viii. dose-dependence of the overall spectrum.there is considerable controversy in the literature concerning the nature of x-ray-induced specific-locus mutations in various experimental organisms. to investigate this problem in neurospora crassa a series of experiments (webber and de serres, 1965) was performed to study the induction-kinetics of x-ray-induced mutation in the adenine-3 (ad-3) region of a two-component heterokaryon (h-12). subsequent genetic analyses (de serres, 1989a,b,c, 1990a), on a series of 832 mutants recovered in these ...19911824716
neurospora crassa clock-controlled genes are regulated at the level of transcription.although an extensive number of biological processes are under the daily control of the circadian biological clock, little is known about how the clock maintains its regulatory networks within a cell. an important aspect of this temporal control is the daily control of gene expression. previously we identified two morning-specific genes that are regulated by the clock through daily control of gene expression (j. loros, s. denome, and j.c. dunlap, science 243:385-388, 1989). we have now introduce ...19911824715
in vitro inhibition of stable 1,3-beta-d-glucan synthase activity from neurospora crassa.glucan synthase activity of neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, ph 7.4, containing 5 mm edta, 1 mm phenylmethylsulfonylfluoride, 200 mm inorganic phosphate, 10 microm gtp, 1 mm dtt, 10 mm sodium fluoride, and 600 mm glycerol. resulting activity was partially purified by sucrose gradient density sedimentation ...19911669437
nucleotide sequence of a neurospora crassa ribosomal protein gene. 19902147995
nucleotide sequence of the exons of the large subunit rrna of neurospora crassa mitochondria. 19901701879
genes expressed during conidiation in neurospora crassa: molecular characterization of con-13.asexual development in neurospora crassa proceeds through a series of discrete morphological stages that culminate in the production of dormant spores called conidia. changes in the pattern of gene expression parallel the morphological transformations associated with conidiation. as a prerequisite to the analysis of developmental gene expression in n. crassa, several genes of unknown function that are preferentially expressed during conidiation were isolated [berlin and yanofsky, mol. cell. biol ...19902148538
glutamine metabolism and cycling in neurospora crassa.[this corrects the article on p. toc in vol. 54.].199016350247
5-benzylidene pyrrolones. iv--synthesis and antifungal activity of some 5-benzylidene derivatives of 1, order to investigate the antifungal activity of pyrrolones, the synthesis of derivatives of 5-benzylidene-1,2,-dimethyl-3-carbethoxy-pyrrol-4-one was achieved by a one-step reaction. the condensation reaction was applied to the above-mentioned heterocycle and seven substituted benzaldehydes were obtained providing the entitled compounds, of which six are original. they have preferential e configuration. antifugal data against neurospora crassa in comparison with ketoconazole showed that many ...19902151022
generation of new functional mutant alleles by premeiotic disruption of the neurospora crassa am the further analysis of a cross in which the mis-sense allele, am3, of the neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the rip process whereas the am3 allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. the am3 allele had also acquired a new hindiii restriction site. it had no detectab ...19902150348
cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium.cytosolic free calcium ([ca2+]c) has been measured in the mycelial fungus neurospora crassa with ca2(+)-selective microelectrodes. the mean value of [ca2+]c is 92 +/- 15 nm and it is insensitive to external ph values between 5.8 and 8.4. simultaneous measurement of membrane potential enables the electrochemical potential difference for ca2+ across the plasma membrane to be estimated as about -60 kj.mol-1-a value that cannot be sustained either by a simple ca2(+)-atpase, or, in alkaline condition ...19902147513
regulation of branched-chain amino acid biosynthesis in neurospora crassa: cloning and characterization of the leu-1 and ilv-3 genes.the genes coding for the branched-chain amino acid biosynthetic enzymes comprise an integrated regulatory system. the expression of the several structural genes coding for enzymes of the isoleucine-valine and leucine pathways is controlled in parallel by the positive-acting regulatory gene, leu-3. the leu-1 and ilv-3 genes, coding for beta-isopropyl-malate dehydrogenase and aceto-hydroxyacid synthase, respectively, were cloned from a cosmid library. restriction fragment length polymorphism analy ...19901980003
studies on the sedimentation behavior of the neurospora crassa plasma membrane h(+)-atpase synthesized in vitro and integrated into homologous microsomal membranes.rna transcripts that encoded the neurospora crassa plasma membrane h(+)-atpase (pma+), a polytopic integral membrane protein, and the pma+344, a truncated pma+ with the amino terminal 344 amino acids, were translated in a n. crassa in vitro system. the microsomal membranes integrated products were insensitive to extraction by na2co3 (ph 11.5). the velocity sedimentation behavior of the in vitro synthesized pma+ were examined under various conditions. the pma+ migrated on linear sucrose gradients ...19902148269
expression of tyrosinase in vegetative cultures of neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene.wild-type neurospora crassa, strain singapore, was transformed with a n. crassa metallothionein promoter/protyrosinase fusion gene. transformants produced tyrosinase during vegetative growth, as determined by western analyses and activity assays. this is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosina ...19902147580
characterization of a novel plasmid-like element in neurospora crassa derived mostly from the mitochondrial dna.we have identified a plasmid-like element within mitochondria of neurospora crassa strain stp-b1. it is derived from the ecori-4 and ecori-6 regions of the mitochondrial dna, and an additional 124 bp dna segment of unknown origin. the plasmid dna consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. the abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. an unusually large number of palindromes capable o ...19902145549
molecular cloning of the l-amino-acid oxidase gene from neurospora crassa.the addition of d-phenylalanine to starved cultures of neurospora crassa leads to de novo synthesis of l-amino-acid oxidase. poly(a) rna from d-phenylalanine-treated mycelium was therefore used to generate a cdna library which was subsequently screened by hybrid-selected translation. a positive l-amino-acid oxidase clone served as a probe to isolate the complete gene from a genomic library of n. crassa. the nucleotide sequence obtained revealed an open reading frame coding for a protein of 695 a ...19902145270
tad, a line-like transposable element of neurospora, can transpose between nuclei in heterokaryons.the tad transposon of neurospora crassa appears to be a line-like element with very restricted distribution within the genus neurospora. when forced heterokaryons were constructed between strains which did and did not contain tad, the nuclei of the naive nuclear type rapidly acquired tad elements. the elements acquired by naive nuclei are active, since they can pass tad to other naive nuclei in subsequent heterokaryons. when heterokaryons are passaged by serial transfer, the load of acquired tad ...19902174012
heat shock response in neurospora crassa: purification and some properties of hsp 80.the heat shock response of neurospora crassa was investigated. a 80-kilodalton heat shock protein (hsp 80) was purified to near homogeneity from heat-shocked mycelial extracts employing ammonium sulphate fractionation, gel filtration, and ion-exchange and affinity chromatography. it was observed to migrate as a single band on one-dimensional sodium dodecyl sulphate--polyacrylamide gels, with a molecular mass of approximately 83 kilodaltons (kda). on two-dimensional gels it resolved into four pol ...19902148482
cpc-2, a new locus involved in general control of amino acid synthetic enzymes in neurospora neurospora crassa starvation for single amino acids leads to derepression of enzymes in many amino acid synthetic pathways. regulation occurs at the level of transcription via "general amino acid (or cross-pathway) control". in this paper a new regulatory gene, cpc-2, is described that specifies a positive, trans-acting effector involved in this control. this gene, located on linkage group vii, was identified by a recessive mutation, u142, which results in sensitivity for two amino acid analo ...19902147403
the nucleotide excision repair epistasis group in neurospora crassa.dna repair mutants in eucaryotes are normally assigned to three epistasis groups. each epistasis group represents a "pathway" for dna repair. the pathways are commonly designated (1) nucleotide excision repair, (2) recombination repair and (3) mutagenic repair. an excision repair epistasis group has been established in neurospora and the mutants assigned to this group should be limited in their ability to excise pyrimidine dimers and other bulky lesions from dna. using a pyrimidine dimer-specifi ...19902147402
expansion and contraction of the nucleolus organizer region of neurospora: changes originate in both proximal and distal segments.previously we have shown that the nucleolus organizer region (nor) of neurospora crassa changes size frequently during the premeiotic portion of the sexual phase. here, we have investigated whether these changes in size originate only in specific regions of the nor, or are distributed throughout the nor. in two special strains of neurospora, the nor was divided into proximal and distal segments. in the first, the nor was divided by a translocation breakpoint and, in the second, the nor was divid ...19902147160
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. vii. genetic lesions resulting in gene/point mutations at the ad-3b locus have different dose-response relationships.genetic characterization of x-ray-induced ad-3 mutants, induced in a two-component heterokaryon (h-12) of neurospora crassa, has been performed to determine genotype, patterns of allelic complementation, and leakiness, and to distinguish gene/point mutations from multilocus deletions and multiple locus mutations (de serres, 1989c, 1990a). the array of genotypes in the classes and subclasses in the spectrum of ad-3 mutants induced by a mutagenic agent constitute its mutagenicity profile; for x-ra ...19902145510
cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of neurospora crassa.carotenoid biosynthesis is regulated by blue light during growth of neurospora crassa mycelia. we have cloned the al-1 gene of n. crassa encoding the carotenoid-biosynthetic enzyme phytoene dehydrogenase and present an analysis of its structure and regulation. the gene encodes a 595-residue polypeptide that shows homology to two procaryotic carotenoid dehydrogenases. rna measurements showed that the level of al-1 mrna increased over 70-fold in photoinduced mycelia. transcription run-on studies i ...19902144609
molecular cloning and analysis of the scon-2 negative regulatory gene of neurospora crassa.the sulfur regulatory system of neurospora crassa is composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control of scon+ (sulfur controller) negative and cys-3+ positive regulatory genes. in scon-1 (previously designated sconc) and scon-2 mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. the scon-2+ gene was cloned by sib selection screening of a cosmi ...19901975945
primary structure and expression of a nuclear-coded subunit of complex i homologous to proteins specified by the chloroplast genome.a 31-kda subunit of complex i from neurospora crassa, of nuclear origin, was cloned. the precursor polypeptide (33 kda) could be efficiently expressed in an in vitro system for transcription and translation. the processing of the precursor to the mature protein was also obtained in vitro. an open reading frame coding for a precursor protein of 283 amino acids (32247 da) was found by dna sequencing. the predicted primary structure shows significant homology with proteins made in chloroplast. this ...19902145832
the reaction of cn- with the binuclear copper site of neurospora tyrosinase: its relevance for a comparison between tyrosinase and hemocyanin active sites.the equilibrium and the kinetics of the reaction between neurospora crassa tyrosinase and cyanide have been studied. cyanide reacts with the binuclear copper active site of the protein competitively with respect to dioxygen and displaces the metal ions. this process occurs stepwise and involves transient intermediates containing mononuclear cu(i) sites. the reaction mechanism proved to be the same as described earlier for molluscan and arthropodan hemocyanins, which share with tyrosinase the sam ...19902145978
identification of the major cytoplasmic regions of the neurospora crassa plasma membrane h(+)-atpase using protein chemical techniques.the transmembrane topography of the neurospora crassa plasma membrane h(+)-atpase has been investigated using purified, reconstituted components and direct protein chemical techniques. reconstituted proteoliposomes containing h(+)-atpase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the h(+)-atpase as recently described (hennessey, j.p., jr., and scarborough, g. (1990) j. biol. c ...19902144525
nucleotide sequence and dna recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of neurospora crassa.the nitrogen regulatory circuit of neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. this set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. the complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. the alc gene ...19902148685
gene sequence and analysis of hsp30, a small heat shock protein of neurospora crassa which associates with mitochondria.hsp30 is a small heat shock protein of neurospora crassa which earlier studies suggested may associate with mitochondria during cellular heat shock. we show here that the association of hsp30 with mitochondria is reversible and that hsp30 dissociates after cells are returned to normal temperature. we sequenced the gene for hsp30 and defined its transcript by s1 nuclease analysis and cdna sequencing. the gene apparently is present in the genome as a single copy, and it contains no introns. the en ...19902144284
sorting pathways of mitochondrial inner membrane proteins.two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. in the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. they become proteolytically processed, interact with the 60-kda heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. the sorting of subunit 9 of neurosp ...19902145157
restricted activation of general amino acid control under conditions of glutamine limitation in neurospora neurospora crassa limitation for single amino acids normally results in increased formation of enzymes required for amino acid synthesis via 'general amino acid control'. glutamine limitation, however, led to comparatively low and delayed derepression of enzyme synthesis. nitrate reductase activity increased steeply under these conditions confirming that de novo protein synthesis could occur. derepression levels were unaffected by addition of glutamine-derived metabolites. only small and dela ...19902148607
glutamine metabolism and cycling in neurospora crassa.evidence for the existence of a glutamine cycle in neurospora crassa is reviewed. through this cycle glutamine is converted into glutamate by glutamate synthase and catabolized by the glutamine transaminase-omega-amidase pathway, the products of which (2-oxoglutarate and ammonium) are the substrates for glutamate dehydrogenase-nadph, which synthesizes glutamate. in the final step ammonium is assimilated into glutamine by the action of a glutamine synthetase (gs), which is formed by two distinct ...19902145504
behavior of the [mi-3] mutation and conversion of polymorphic mtdna markers in heterokaryons of neurospora crassa.we have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtdna markers in heterokaryotic cultures of neurospora crassa. previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of ecori-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. we have confirmed this observation and determined by dna sequence analysis that the insertion in the ecori-5 fragment corresponds precisely to an optional intron that ...19901977658
isolation and characterization of a neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase.we report the isolation and characterization of a neurospora crassa glutamine synthetase (gs) mutant altered in one of the two polypeptides (gs alpha) of this enzyme. we used the gln-1br8 mutant strain that synthesizes only the gs alpha monomer and lacks the gs beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-dl-methionine-sr-sulfoximine (alpha-me-mso), an inhibitor of gs activity. the gs activity of the gln-1br8;alpha-me-msor strain drastically reduced its ...19901975579
nucleotide sequence of a full-length cdna coding for the mitochondrial precursor protein of the beta-subunit of f1-atpase from neurospora crassa. 19902144339
effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes: a study on mycelial and wall-less phenotypes of the fz; sg; os-1 ('slime') triple mutant of neurospora crassa.the production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain rcp-3) of a cross 'slime' x wild type. the unrelated stable 'slime' strain fgsc 1118 was also studied. the synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-cat ...19902262786
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. vi. induction kinetics of gene/point mutations, multilocus deletions and multiple-locus mutations.genetic fine-structure analysis of x-ray-induced specific-locus mutants in the ad-3 region of two-component heterokaryons of neurospora crassa has shown that gene/point mutations, multilocus deletions and multiple-locus mutations are induced. when the dose-response curves for these classes of ad-3 mutants were plotted, it was demonstrated that x-ray-induced gene/point mutations (ad-3r) increased linearly with x-ray dose and x-ray-induced multilocus deletions increased as the square of the x-ray ...19902143556
biochemical, genetic and ultrastructural defects in a mitochondrial mutant (er-3) of neurospora crassa with senescence phenotype.the structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant er-3 of neurospora crassa are described. the mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. however, the total whole cell respiration rate of the mutant was 33% gre ...19902169558
analysis of conventional and in vitro generated mutants of nmr, the negatively acting nitrogen regulatory gene of neurospora crassa.the nmr gene is the major negative regulatory gene in the nitrogen control circuit of neurospora crassa, which, together with positive regulatory genes, governs the expression of multiple unlinked structural genes of the circuit. possible functional domains of the nmr protein were investigated by mutational analyses using three different approaches. first, the polymerase chain reaction was used to clone the nmr locus from two conventional mutants, v2m304 and ms5, and the mutant amino acid codons ...19902148799
distant upstream regulatory sequences control the level of expression of the am (gdh) locus of neurospora crassa.we have constructed deletions in the 5' noncoding sequences of the cloned neurospora crassa am gene. vectors with a truncated fragment of the am gene were used in transformation experiments to introduce the deletions into the chromosome by homologous recombination. analysis of glutamate dehydrogenase (gdh) expression by enzyme assay and immunoblots, as well as northern and dot blots of poly (a)+ rna, in the deletion strains indicates that there are two upstream regulatory sequences that control ...19902147126
the cellulase complex of neurospora crassa: activity, stability and release.the temperature and ph optima, and the temperature and ph stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus neurospora crassa were investigated. the effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of cellulase complex were also examined. for the different enzymes of the complex, activity maxima occurred between ph 4.0 and 7.0, with ph 5.0 being close to optimal for stability of all. temperature optim ...19902146364
assembly of nadh: ubiquinone reductase (complex i) in neurospora mitochondria. independent pathways of nuclear-encoded and mitochondrially encoded subunits.nadh:ubiquinone reductase, the respiratory chain complex i of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one fmn, probably one internal ubiquinone and at least four iron-sulphur clusters. we are studying the assembly of the enzyme in neurospora crassa. the flux of radioactivity in cells that were pulse-labelled with [35s]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. la ...19902141652
a polypeptide of 59 kda is associated with bundles of cytoplasmic filaments in neurospora crassa.complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus neurospora crassa. they were enriched by differential centrifugation and purified by permeation chromatography. the filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. the main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kda (p59nc), which represents 4-5% of the total n. crassa protein ...19902141976
intracellular sodium content of a wall-less strain of neurospora crassa and effects of insulin: a 23na-nmr study.23na-nmr has been used to investigate some factors influencing the sodium content of a wall-less strains of neurospora crassa. the shift reagent tm(dotp)h2(nh4)3 proved useful for this purpose, while several other reagents, previously used by others, were found to be unsuitable for use with these cells. when the cells were grown, washed and resuspended in medium containing sodium (25.3 mm), the intracellular sodium concentration was calculated to be 11.9 +/- 1.4 mm. this value rose within two mi ...19902142438
processing of precursor proteins by plant mitochondria.precursor proteins from neurospora crassa were correctly processed by a matrix extract from vicia faba and cauliflower mitochondria. processing yielded mature protein of the same molecular mass as mature neurospora protein. the processing activity has two components. one is antigenically related to and of the same molecular mass as the processing enhancing protein of neurospora. the second component was not recognized by antibody to the matrix processing protease from neurospora mitochondria. th ...19902140932
duplication-induced mutation of a new neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.a cloned neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic dna and the normally located gene. such changes are typical of the duplication-induced premeiotic disruption (the rip effect) first described by selker et al. (e. u. sel ...19902140429
molecular cloning and characterization of alc the gene encoding allantoicase of neurospora crassa.purines can be utilized as a secondary nitrogen source by neurospora crassa during conditions of nitrogen limitation. the expression of purine catabolic enzymes is governed by the nitrogen regulatory circuit and requires induction by uric acid. the major positive-acting nitrogen regulatory gene, nit-2, turns on the expression of the purine catabolic enzymes, which may also be subject to negative regulation by a second control gene, nmr. we have cloned alc, the structural gene which encodes allan ...19901978237
chemical state of the cysteine residues in the neurospora crassa plasma membrane h(+)-atpase.the plasma membrane h(+)-atpase of neurospora crassa was treated with 5,5'-dithiobis(2-nitrobenzoate) to determine its cysteine content and with 2-nitro-5-thiosulfobenzoate to determine its cystine content. six and seven mol of thiols/mol of h(+)-atpase were detected in the 5,5'-dithiobis(2-nitrobenzoate) and 2-nitro-5-thiosulfobenzoate reactions, respectively, indicating that 6 of the 8 cysteine residues in the molecule are present as free cysteines and that 2 are present in disulfide linkage. ...19902139659
blue light photoreception in neurospora circadian rhythm: evidence for involvement of the flavin triplet state.the mechanism of the photoreceptor acting on the circadian conidiation rhythm of neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) phase shifts were not abolished by removal of oxygen prior to illumination. (3) oxygen inhibited phase shifts when introduced into the cultures after light treatment. it is proposed that the blue light photoreceptor for the circadian clock of neurospora crassa acts (1 ...19902367558
development of thermotolerance in neurospora crassa by heat shock and other stresses eliciting peroxidase induction.hyperthermia, cdcl2, sodium arsenite, and h2o2 led to the rapid appearance of high levels of peroxidase in neurospora crassa cultures and induced tolerance toward normally lethal temperatures in 60-h-old colonies. intracellular superoxide dismutase levels did not correlate with the development of thermotolerance.19902139653
the genetics of polyamine synthesis in neurospora mutations of the polyamine pathway of neurospora crassa fell into three categories. the majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. one mutation, jp100, defining the new spe-2 locus, eliminated s-adenosyl-methionine decarboxylase and led to putrescine accumulation. revertants of this mutation suggested that the locus encodes the enzyme. two other mutations, lv105 and jp120, defined a third locus, spe-3. strains with these mutations also accumulate ...19902139316
characterization of telomere dna from neurospora crassa.the nucleotide sequence of the telomere at the right end of linkage group v (vr) in the standard or23-iv-a strain of the filamentous fungus, neurospora crassa, reveals the following features. at the chromosome terminus, tandem repeats of the hexanucleotide ttaggg are present. immediately centromere-proximal to the simple sequence repeat is a more complex element called pogo that is reiterated 5-10 times in the genomes of various neurospora strains. the element possesses several features characte ...19901971801
light-induced dephosphorylation of a 33 kda protein in the wild-type strain of neurospora crassa: the regulatory mutants wc-1 and wc-2 are abnormal.light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of neurospora crassa. the regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. because the wc genes have previously been implicated in photodifferentiation (f. degli innocenti and v. e. a. russo, genetic analysis of blue light-induced responses in neurospora crassa, in h. senger (ed.), blue light effects in biological systems, springer- ...19902140412
very low atp/adp ratios with aging of the natural death senescence mutant of neurospora crassa.the natural death (nd) mutant of the fungus neurospora crassa, unlike the wild-type, undergoes an aging process, which leads to the cessation of growth. it is shown here that the atp/adp ratio of the mutant declines with age to about 3:1 whereas other strains of neurospora in the same growth medium maintain ratios of about 8 to 9:1. the decline in atp/adp ratio is not caused by the cessation of growth of the mutant. the results suggest, rather, that the cessation of growth may be caused, in part ...19902139154
relationship of vector insert size to homologous integration during transformation of neurospora crassa with the cloned am (gdh) gene.we used lambda and plasmid vectors containing the am+ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. a total of 199 transformants were examined with the potential to yield am+ transformants by homologous recombination. when we used vectors that had 9.1 kb of homology with the chromosomal dna, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a pl ...19902157957
dna methylation and chromatin structure: a view from understanding of the function and control of dna methylation in eukaryotes has been elusive. studies of neurospora crassa have led to a model that accounts for the chromosomal distribution of methylation and suggests a basic function for dna methylation in eukaryotes.19902139257
determination of the inactivating alterations in two mutant alleles of the neurospora crassa cross-pathway control gene cpc-1.cpc-1 is the locus specifying what is believed to be the major trans-activating transcription factor that regulates expression of amino acid biosynthetic genes subject to cross-pathway control in neurospora crassa. mutants altered at this locus are incapable of the global increase in gene expression normally seen in response to amino acid starvation. using polymerase chain reaction methodology we have cloned and sequenced the inactive mutant allele, cpc-1 (cd15). the cpc-1 (cd15) mutation was fo ...19902138111
synaptic adjustment of inversion loops in neurospora crassa.heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. this synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cm long loops.19902138110
nit-2, the major nitrogen regulatory gene of neurospora crassa, encodes a protein with a putative zinc finger dna-binding domain.the nitrogen regulatory circuit of neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. the positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. the complete nucleotide sequence of the nit-2 gene was determined. the nit-2 mrna is 4.3 kilobases long and has a long ...19902137552
cis,cis-cyclohexane 1,3,5-triol polyphosphates release calcium from neurospora crassa via an unspecific ins 1,4,5-p3 receptor.we investigated the effects of new inositol 1,4,5-trisphosphate analogues on the release of ca2+ from isolated vacuoles of neurospora crassa. tri-o-butyryl-inositol 1,4,5-trisphosphate and a set of cis,cis-cyclohexane 1,3,5-triol bis-(cht-p2) and trisphosphates (cht-p3) gave an increase in free ca2+ as measured directly with fura-2, a ca2(+)-chelator. however, inositol 1,4-bisphosphate, 6-o-palmitoyl-inositol 4,5-bisphosphate and trans-cyclohexane 1,2-diol bisphosphate (trans chd-p2) did not ind ...19902154977
the range of amino acids whose limitation activates general amino-acid control in neurospora crassa.several amino-acid synthetic enzymes, belonging to arginine, glutamine, leucine, lysine and phenylalanine biosynthesis, respectively, were investigated under conditions of reduced availability of any one of 16 out of the 20 amino acids represented in proteins. the enzymes showed simultaneous derepression under each condition, albeit to different degrees. derepression was abolished and the remaining basal enzyme levels reduced by mutations at the cpc-1 locus which governs general amino-acid contr ...19902138581
cellulase production by neurospora crassa: purification and characterization of cellulolytic studies on cellulase production by the cell-1 mutant of neurospora crassa, eight enzymes (three exoglucanases, four endoglucanases, and one beta-glucosidase) were identified and characterized by gel filtration, ion exchange chromatography, and chromatofocusing. after purification, each of the proteins ran as a single band in polyacrylamide gel electrophoresis, using both native and denaturing gels. the molecular weights of the proteins were found to be between 70,000 and 22,000 daltons, and a ...19901368541
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