TitleAbstractYear(sorted ascending)
levels of sulfhydryls and disulfides in proteins from neurospora crassa conidia and mycelia.proteins extracted with 6 m guanidine at 90 degrees c from conidia (asexual spores) of neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14c]acetic acid. the total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. the level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by t ...19836226648
stoichiometry of h+/amino acid cotransport in neurospora crassa revealed by current-voltage analysis.coupling of ions to the uptake of neutral and basic amino acids via a general amino acid transport system (system ii), was studied in a mutant of neurospora crassa (bat mtr) which lacks other transport systems for these solutes. all amino acids tested--including ones bearing no net charge--elicited rapid membrane depolarization, as expected for ion-coupled transport. (since amino acid transport in neurospora is not dependent on extracellular na+ or k+, the associated ion is presumed to be h+.) a ...19836226314
isolation and characterization of the neurospora crassa endoplasmic reticulum.the endoplasmic reticulum from neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. after differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane h+-atpase. however, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. the lighter membrane f ...19836311800
repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities in neurospora crassa.two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type neurospora crassa and purified. the two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. this enzyme had the same electrophoretic mobility as repressible acid phosphatase. the enzyme designated repre ...19836311798
comparison of the cytotoxic and mutagenic effects of selected mutagens on excision repair-sufficient and -deficient ad-3 mutants of neurospora crassa.the excision repair-deficient genetic marker uvs-2 was crossed into the tester strains n23 and n24 of neurospora crassa. comparison was made among the effects of selected mutagens on a repair-sufficient strain (n23 or n24) and a repair-deficient strain (n23 uvs-2 or n24 uvs-2) with regard to cell killing and induction of reverse mutation from adenine dependence to adenine independence. methyl methanesulfonate (mms), ethyl methanesulfonate (ems), 1,2,7,8-diepoxyoctane (deo), n-methyl-n'-nitro-n-n ...19836226873
neurospora crassa mutants deficient in asparagine synthetase.neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. recombination analysis with strain s1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group v. in vitro assays with a heterokaryon indicated that the mutation was dominant. thermal instability of cell ex ...19836137480
segregation of heterogeneous rdna segments during demagnification of a neurospora crassa strain possessing a double nucleolar organizer.we have produced a duplication strain of neurospora crassa, dpar33-pr-6, which contains two cytologically visible nucleoli (a dno or double nucleolar organizer strain). when freshly generated, this strain has approximately twice the number of rrna cistrons found in the parental (single nucleolar organizer) strains. after several serial propagations, there is a marked reduction in rrna cistron number, approximating that of the sno parental strains. this reduction in rrna cistrons ("demagnificatio ...198324173419
leucine biosynthesis in yeast : identification of two genes (leu4, leu5) that affect α-isopropylmalate synthase activity and evidence that leu1 and leu2 gene expression is controlled by α-isopropylmalate and the product of a regulatory gene.tetrad analysis indicates that α-isopropylmalate synthase activity of yeast is determined by two separate genes, designated leu4 and leu5. leu4 is identified as a structural gene. leu5 either encodes another α-isopropylmalate synthase activity by itself or provides some function needed for the expression of a second structural gene. the properties of mutants affecting the biosynthesis of leucine and its regulation suggest that the expression of leu1 and leu2 (structural genes encoding isopropylm ...198324173418
epistatic grouping of repair-deficient mutants in neurospora: comparative analysis of two uvs-3 alleles, uvs-6 and their mus double mutant strains.the nuclease halo mutant, nuh-4, of neurospora crassa was identified conclusively as an allele of uvs-3, a gene involved in error-prone dna repair. like uvs-3, nuh-4 showed spontaneous mutator effects, and any previous contradictory findings were found to be due to newly arisen mutants. in normal strains the two alleles are noncomplementing and indistinguishable for sensitivity to uv and methyl methanesulfonate (mms). like uvs-3, nuh-4 lacked secretion of the extracellular enzyme, dnase a, a ca( ...198317246153
cell wall composition of neurospora crassa under conditions of copper toxicity.the mycelia of neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. the cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. although all the glucosamine of the cell wall of control cultures could be released withi ...198316346385
two-dimensional electrophoresis of plasma membranes, showing differences among wild-type and abnormal ascospore mutant strains of neurospora crassa.plasma membranes isolated from vegetative cultures of wild-type neurospora crassa were analyzed by two-dimensional electrophoresis, followed by staining with silver nitrate to visualize proteins and fluorescein-labeled concanavalin a to visualize glycosylated subunits. mycelial plasma membranes from strains carrying mutations affecting ascospores were also analyzed. two of the mutant strains were shown to have aberrant two-dimensional membrane subunit patterns. the correlation of these abnormali ...19836224773
genetic analysis of phototropism of neurospora crassa perithecial beaks using white collar and albino mutants.positive phototropism of perithecial beaks in the fungus neurospora crassa has been demonstrated. the effect was shown to be mediated by blue light. when mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. however, when wild type, albino-1, albino-2, or albino-3 strains were used as the protope ...198316663152
biosynthesis of glycogen in neurospora crassa. purification and properties of the branching enzyme.a branching enzyme was extracted from the mycelia of neurospora crassa and was purified to electrophoretic homogeneity by procedures including deae-sephacel column chromatography, 6-aminohexyl-sepharose 4b column chromatography and gel filtration on toyopearl hw-55s. the final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. the molecular weight of this enzyme was estimated to be 80,000 by elect ...19836226652
isolation of a bifunctional domain from the pentafunctional arom enzyme complex of neurospora proteolysis of the arom enzyme complex of neurospora crassa by trypsin or subtilisin yielded a stable fragment of mr 68000. this fragment, which was purified by two-dimensional polyacrylamide-gel electrophoresis, was shown by activity staining to contain the shikimate dehydrogenase active site, and by substrate labelling with 3-dehydroquinate and nab3h4 to contain the 3-dehydroquinase active site. the fragment thus constitutes a bifunctional domain containing the two enzymic activities t ...19836225423
effects of mg2+ ions on the plasma membrane [h+]-atpase of neurospora crassa. ii. kinetic studies.the rate of atp hydrolysis by the neurospora plasma membrane [h+]-atpase has been measured over a wide range of mg2+ and atp concentrations, and on the basis of the results, a kinetic model for the enzyme has been developed. the model includes the following three binding sites: 1) a catalytic site at which mgatp serves as the true substrate, with free atp as a weak competitive inhibitor; 2) a high affinity site for free mg2+, which serves to activate the enzyme with an apparent k1/2 (termed kmga ...19836223037
effects of mg2+ ions on the plasma membrane [h+]-atpase of neurospora crassa. i. inhibition by n-ethylmaleimide and trypsin.we have shown previously (brooker, r.j., and slayman, c.w. (1982) j. biol. chem. 257, 12051-12055; brooker, r. j., and slayman, c. w. (1983) j. biol. chem. 258, 222-226) that the plasma membrane [h+]-atpase of neurospora crassa is inhibited by n-ethylmaleimide (nem), which reacts at an essential nucleotide-protectable site on the mr = 104,000 polypeptide. the present study demonstrates that mg2+ has a biphasic effect on nem inhibition. at low concentrations (0.01-0.1 mm, mg2+ decreases the sensi ...19836223036
compartmentation of spermidine in neurospora crassa.the polyamines putrescine, spermidine, and spermine are multivalent cations that bind to anionic cell constituents such as nucleic acids. their distribution between free and bound states within the cell is not known. such knowledge would be important in relation to the negative control of polyamine synthesis. we report a tracer experiment in which [14c]ornithine was added to logarithmically growing neurospora crassa mycelia. the amount and the specific radioactivity of the three polyamines there ...19836223033
calmodulin-stimulated cyclic nucleotide phosphodiesterase from neurospora crassa.cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-sepharose affinity chromatography from a soluble extract of neurospora crassa. the phosphodiesterase activity remained bound to the affinity column even in the presence of 6 m urea and could only be eluted by calcium chelation. the enzyme exhibits camp and cgmp phosphodiesterase activities. both activities can be enhanced by calmodulin in a ca2+-dependent manner. stimulation of cyclic nucleotide phosphodiesterase by ca ...19836305427
modification of blue light photoresponses by riboflavin analogs in neurospora crassa.the effect of riboflavin analogs on blue light responses in a riboflavin mutant of neurospora crassa was studied. the analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, acted as photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were ineffective as photoreceptors for the induction of carotenoid synthesis. these results provide addtional evidence implicating a flavin photoreceptor for at least two blue light response ...198316663082
neurospora crassa mutant impaired in glutamine regulation.the final products of the catabolism of arginine that can be utilized as nitrogen sources by neurospora crassa are ammonium, glutamic acid, and glutamine. of these compounds, only glutamine represses arginase and glutamine synthetase. we report here the isolation and characterization of a mutant of n. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glni). this mutant has a greater capacity than the wild type (glns) to accumulate mo ...19836134713
calcium as a branching signal in neurospora crassa.the divalent cation ionophore a23187 was found to induce apical branching in neurospora crassa. optimal effects were obtained by treatment with 0.1 mm ionophore for 30 min. branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. the branching response was ma ...19836304014
the relationship between conidial germination and esterase activities in neurospora crassa.esterase activity in rapidly germinating neurospora conidia was several times higher than the esterase activity in conidia which germinate slowly. starch gel electrophoresis experiments demonstrated the existence of esterase isoenzymes which are specific to the conidia. these isoenzymes completely disappeared during 20 h of conidial germination at 30 degrees c. electron microscopy showed the successive breakdown of electron-dense compounds in storage bodies during conidial germination. these obs ...19836226763
high-performance liquid chromatography for assaying nad glycohydrolase from neurospora crassa conidia.a rapid and sensitive high-performance liquid chromatographic technique was developed to determinate nad glycohydrolase (ec activity from neurospora crassa conidia. the separation of the assay substrate and products was achieved by isocratic reverse-phase chromatography and the peaks were detected by the absorbance at 259 nm. quantities of nad+ and nicotinamide as small as 10 pmol could be measured.19836225349
cell-biology of ageing. 11. the effect of vitamin e, vitamin c and sodium selenite on the ageing syndromes of early senescent mutants of neurospora crassa. 19836224572
co-accumulation of prephenate, l-arogenate, and spiro-arogenate in a mutant of neurospora.a mutant strain of neurospora crassa blocked in each of the initial steps of tryptophan, tyrosine, and phenylalanine biosynthesis was previously shown to accumulate and secrete prephenate and l-arogenate (jensen, r.a., zamir, l.o., st. pierre, m., patel, n., and pierson, d.l. (1977) j. bacteriol. 132, 896-903). we now report the co-accumulation of yet another compound which was identified (zamir, l.o., tiberio, r., jung, e., and jensen, r.a. (1982) j. biol. chem. (1983) 258, 6486-6491) as the la ...19836222045
isolation and structure determination of a novel spiro-gamma-lactam, spiro-arogenate.the eucaryotic microorganism, neurospora crassa, is able under specified conditions (zamir, l.o., jung, e., and jensen, r.a. (1982) j. biol. chem. 258, 6492-6496) to synthesize a cyclohexadienyl derivative of prephenic acid having the novel structure of a spiro-gamma-lactam. this l-gamma-(spiro-4-hydroxy-2,5-cyclohexadienyl)-pyroglutamate is herein given the trivial name, spiro-arogenate, to indicate its close relationship to the amino acid, l-arogenate. spiro-arogenate is quantitatively convert ...19836222044
control points in neurospora crassa nuclear division cycle: different effects of the inhibition of protein accumulation.the correlation between protein synthesis and the nuclear division cycle in neurospora crassa hyphae was studied by inhibiting protein accumulation by two different experimental procedures: (1) starvation for lysine in a lysine-requiring mutant (lys-1); and (2) addition of cycloheximide. lysine starvation in a lys-1 strain of n. crassa quickly blocked the nuclear division cycle and nuclei accumulated in g1 phase, as judged by their dna content. after re-addition of lysine to starved cultures, a ...19836224804
use of a temperature-sensitive, protoplast-forming neurospora crassa strain for the detection of antifungal antibiotics.protoplasts of the temperature-sensitive osmotic-1 mutant of neurospora crassa grew and divided as cell wall-less cells when incubated under certain conditions at 37 degrees c. each protoplast regenerated cell wall and formed a mycelium when the temperature was shifted to 22 degrees c. cell wall regeneration, but not cell growth, was prevented by the inhibition of cell wall assembly functions. thus, the inhibition of cell wall regeneration could serve as an indicator of the mode of action of ant ...19836223580
isolation and characterization of light-insensitive mutants of neurospora part of a genetic analysis of blue light photoreception in neurospora, three mutants were isolated that do not exhibit photosuppression of circadian conidiation, i.e., they show periodic conidiation in constant light. the mutations have been given the designations lis-1, lis-2 and lis-3 ("light insensitive"). the three mutations segregate as single nuclear genes, are nonallelic and are recessive to wild type in heterokaryon tests. the linkage groups of the mutations are as follows: lis-1, i; ...19836222936
cell wall assembly of neurospora crassa: lack of evidence for preexisting cell wall acting as primer or template.cell wall formation of neurospora crassa and other filamentous fungi involves the apical extension of preexisting cell wall in a complex assembly sequence; however, it is not known if preexisting wall participates in the formation of new cell wall. it was found that temperature-sensitive protoplasts which lack detectable preexisting wall form cell wall upon a shift to a permissive temperature. similarly, temperature-sensitive colonial mutants form morphologically normal cell wall directly from p ...19836220935
changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of neurospora crassa.growth of a choline requiring auxotroph of neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. in these lipids, choline depriva ...19836219706
interrelationships in trace-element metabolism in metal toxicities in nickel-resistant strains of neurospora crassa.three different ni2+-resistant strains of neurospora crassa (nir1, nir2 and nir3) have been isolated. all are stable mutants and are fourfold more resistant to ni2+ than the parent wild-type strain. nir1 and nir2 are also sixfold more resistant to co2+, whereas nir3 is only twice as resistant to co2+; the former two are also twofold more resistant to zn2+, but nir3 is not. these three strains also differ in sensitivity to cu2+. toxicities and concomitant accumulation patterns of ni2+, co2+ and c ...19836223632
transfer of proteins into mitochondria. precursor to the adp/atp carrier binds to receptor sites on isolated mitochondria.the precursor form of neurospora crassa mitochondrial adp/atp carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. if the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. upon reestablishment of the membrane potential, the bound precursor is imported. this occurs without dissociation of the bound precursor from the mitochondrial surface. we conclude that the bindin ...19836300074
kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate.the herbicide glyphosate (n-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (epsp) synthase activity of the purified arom multienzyme complex from neurospora crassa. inhibition of the epsp synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with k(i) 1.1 microm, and uncompetitive with respect to shikimate-3-phosphate. the kinetic patterns are consistent with a compulsory order sequential mechanism in which either ...198311968207
structural and functional evidence for multiple channel complexes in the outer membrane of neurospora crassa mitochondria.the outer membrane of mitochondria contains proteins that form channels called vdac (voltage-dependent anion-selective channels). two independent lines of evidence suggest that these channels occur in specific complexes in the outer membrane of neurospora mitochondria. electron microscopic images of these outer membranes reveal polymorphic crystalline arrays of putative pores. these arrays can be shown to be interrelated by movement in the membrane plane of a particular rigid channel triplet or ...19836300902
nitrogen source regulates glutamate dehydrogenase nadp synthesis in neurospora crassa.neurospora crassa glutamate dehydrogenase-nadp (ec has a higher activity when mycelium is grown on ammonium or nitrate as nitrogen source than when grown on glutamate or glutamine. quantitative immunoelectrophoresis established that, under all conditions, enzyme activity corresponded to enzyme concentration. isotope incorporation studies demonstrated that the nitrogen source exerts its regulation at the level of de novo enzyme synthesis.19836300039
intracellular localization of neurospora crassa endo-exonuclease and its putative precursor.endo-exonuclease of rapidly growing mycelia of neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. the mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of triton x-100. an inactiv ...19836300036
purification and characterization of the trifunctional beta-subunit of anthranilate synthase from neurospora crassa.the trifunctional beta-subunit of anthranilate synthase complex of neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. the isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on sephacryl s-300 compared with a monomer molecular weight of approximately 84,000 da as determined by sodium dodecyl sulfate-gel electrophoresis. the purified subunit was cl ...19836219992
trans-nuclear action of the nit-2 regulatory gene product and study of two additional nitrogen control genes in neurospora crassa.the nit-2 gene of neurospora crassa is a major regulatory gene for control of nitrogen metabolism. synthesis of the enzyme l-amino acid oxidase requires a functional nit-2 gene product and is also controlled by amino acid induction and nitrogen catabolite repression. electrophoretic variants of l-amino acid oxidase have been employed to demonstrate that in heterokaryons, a nit-2 (+) gene product can turn on the expression of this enzyme in its own nucleus and also in nuclei that possess a nit-2 ...198324173118
the structure of the gene for subunit i of cytochrome c oxidase in neurospora crassa mitochondria.we have sequenced the gene for cytochrome c oxidase subunit 1 (co i) in neurospora crassa mitochondrial dna. the gene is coded by the same strand as the rrna and trna genes. the coding sequence predicts a protein of 557 amino acids, starting with methionine, and ending with asparagine. comparison to the n-terminal amino acid sequence of the mature protein (werner et al. 1980) reveals that the methionine is located at position -2. no other upstream aug codons have been found in frame. the c-termi ...198324173114
reconstitution of adenylate cyclase in neurospora from two components of the enzyme.the atp-mg2+-dependent, guanine nucleotide-stimulated adenylate cyclase of neurospora crassa was shown to be composed of distinct components which, when mixed, can reconstitute a holoenzyme. after brief heat treatment, the adenylate cyclase activity in crude homogenates of neurospora was found to have reduced activity and greatly reduced sensitivity to stimulation by guanyl-5'-yl-imidodiphosphate (gpp(nh)p). gpp(nh)p sensitivity could be restored by addition of a homogenate from a crisp-1 (cr-1) ...19836830259
measurements of cytoplasmic and vacuolar ph in neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy.the nitrogen-15 chemical shift of the n1 (tau)-nitrogen of 15n-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15n-labeled arginine and of the alpha-nitrogens of 15n-labeled alanine and proline were measured in intact mycelia of neurospora crassa to obtain to estimates of intracellular ph. for intracellular 15n-labeled histidine, the n1 (tau)-nitrogen chemical shift was 200.2 ppm. in vitro measurements showed that the che ...19836220738
characterization of an atp-mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from neurospora crassa.a novel adenylate cyclase activity was found in crude homogenates of neurospora crassa. the adenylate cyclase had substantial activity with atp-mg2+ as substrate differing significantly from the strictly atp-mn2+-dependent enzyme characterized previously. additionally, the atp-mg2+-dependent activity was stimulated two- to fourfold by gtp or guanyl-5'-yl-imido-diphosphate (gpp(nh)p). we propose that the atp-mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory comp ...19836219625
modification of the neurospora crassa plasma membrane [h+]-atpase with n,n'-dicyclohexylcarbodiimide.the [h+]-atpase of the neurospora plasma membrane is composed of a single mr = 104,000 polypeptide (b. j. bowman, f. blasco, and c. w. slayman, j. biol. chem. (1981) 256, 12343-12349). the carboxyl-modifying reagent n,n'-dicyclohexylcarbodiimide (dccd) inactivates the atpase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. the rate constant for inactivation at ph 7.5 and 30 degrees c is approximately 1000 m-1 min-1, similar to values reported for the dccd-bin ...19836218168
cyclic amp levels in relation to membrane phospholipid variations in neurospora crassa.the correlation between membrane phospholipid composition and total cyclic amp levels was investigated by using neurospora lipid auxotrophs under various supplementation conditions. the lipid composition of the supplemented cultures was determined, and the intracellular and extracellular cyclic amp levels were measured at various stages of the culture growth. kinetic parameters and the thermostability of adenylate cyclase and of cyclic amp-dependent phosphodiesterase were measured under all supp ...19836307199
re-assessment of ammonium-ion affinities of nadp-specific glutamate dehydrogenases. activation of the neurospora crassa enzyme by ammonium and rubidium ions.the nadp-specific glutamate dehydrogenase of neurospora crassa shows complex interactions with nh4+ ions, characterized by biphasic downwardly convex double-reciprocal plots. these kinetics are explained by the action of nh4+ both as a substrate and, acting at a separate cation-binding site, as an activator. rb+ ions, and to a smaller extent other univalent cations, also activate by acting as analogues of nh4+. previous failure to recognize this effect, which probably also occurs in homologous e ...19836221721
characterization of a 45-kda polypeptide as the precursor of subunit 1 of cytochrome c oxidase in neurospora a previous paper (van 't sant, p., mak, j.f.c. and kroon, a.m. (1981) eur. j. biochem. 121, 21-26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kda) of mitochondrial translation products in neurospora crassa. we presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. here we present conclusive evidence that the 45-kda polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kda polype ...19836299357
[14c]n-ethylmaleimide labeling of the plasma membrane [h+]-atpase of neurospora crassa.treatment of the purified plasma membrane [h+]-atpase of neurospora crassa with the sulfhydryl reagent n-ethylmaleimide (nem) leads to a marked inhibition of atpase activity. several lines of evidence indicate that inhibition is caused by the reaction of nem with a single cysteine residue on the mr = 104,000 polypeptide. (1) inhibition by nem follows pseudo-first order kinetics. (2) mgadp protects against nem inactivation and at the same time decreases the incorporation of [14c]nem into the mr = ...19836217204
on the presence of a heme-binding domain homologous to cytochrome b(5) in neurospora crassa assimilatory nitrate reductase.assimilatory nitrate reductase has been purified with 55% recovery from a neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. it possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b(5). on sds gels, the fraction showed two heme-containing bands of 10 000 and 12 5000 daltons; their amino acid composit ...198316453480
[purification and isolation of the arom aggregates of schizosaccharomyces pombe].the five enzymes that catalyzing steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway are physically associated and have been purified up to 400-fold from schizosaccharomyces pombe. the native arom aggregate has a molecular weight of approx. 140,000-145,000 based on gel filtration, glycerol-density-gradient centrifugation, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. similarities between the s. pombe arom aggregate and that ...19836624143
isolation and properties of the porin of the outer mitochondrial membrane from neurospora crassa. 19836318030
biogenesis of cytochrome c in neurospora crassa. 19836318029
preparation of highly purified mitochondria of neurospora crassa on a percoll gradient.mitochondria isolated from neurospora crassa were purified by centrifugation in a percoll density gradient. enzyme activities and cytochrome differential spectra revealed a high purity of the mitochondria. as compared with a crude mitochondrial fraction the purified mitochondria exhibited a high respiratory activity and a fine adp/o ratio. electrophoresis of nucleic acids demonstrated the absence of cytoplasmic rrna.19836315550
neurospora tyrosinase: structural, spectroscopic and catalytic properties.tyrosinase is a copper containing monooxygenase catalyzing the formation of melanin pigments and other polyphenolic compounds from various phenols. this review deals with the recent progress on the molecular structure of the enzyme from neurospora crassa and the unique features of the binuclear active site copper complex involved in the activation of molecular oxygen and the binding of substrates. the results of the spectroscopic properties of neurospora tyrosinase will also be discussed in the ...19836308414
ribosomal genes of neurospora crassa: constancy of gene number in the conidial and mycelial phases, and homogeneity in length and restriction enzyme cleavage sites within strains.we report the results of experiments which, while not specifically designed to study the possibility of rdna amplification during different developmental stages in the n. crassa life cycle, clearly indicate a relative constancy in the rdna content of conidia (asexual spores) and mycelial cells. we also report the results of restriction enzyme studies which indicate that the neurospora rdna repeat units are homogeneous in length and restriction site pattern within any given neurospora strain. the ...19836227796
uses of arginaseless cells in the study of polyamine metabolism (neurospora crassa). 19836225934
uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of neurospora mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. one strain was deficient for nad-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mm glycerol, but at higher co ...19836222238
a study of the properties of pyruvate kinase isolated from a mutant of neurospora crassa: a comparison with the parental enzyme.1. a mutant of neurospora crassa has been isolated whose pyruvate kinase is twice as active as the wild type enzyme. 2. the purified mutant and the wild type enzymes exhibit similar immunological properties, pi values (6.4) and arrhenius activation energy (11.2 kcal/mol). 3. both the enzymes show hyperbolic saturation kinetics with adp and sigmoidal kinetics with pep. 4. the mutant enzyme displays a higher affinity for pep and a greater extent of cooperativity in binding than the wild type. 5. c ...19836221961
photoinduction of protoperithecia in neurospora crassa by blue light. 19836220417
biosynthetic pathway of mitochondrial atpase subunit 9 in neurospora crassa.subunit 9 of mitochondrial atpase (su9) is synthesized in reticulocyte lysates programmed with neurospora poly a-rna, and in a neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (mr 16,400 vs. 10,500). the rna which directs the synthesis of su9 precursor is associated with free polysomes. the precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. transfer in vitro of the precursor int ...19836219116
vanadate-resistant mutants of neurospora crassa are deficient in a high-affinity phosphate transport system.mutant strains of neurospora crassa have been selected which grow on media containing vanadate, an inhibitor of the plasma membrane atpase. the mutations all map to a single region (designated van) on the left arm of linkage group vii. the van mutants are unable to take up vanadate from the medium and are also deficient in the uptake of phosphate via a derepressible, high-affinity phosphate transport system. in the van mutants, the k(m) for phosphate transport is elevated as much as 35-fold, ind ...19836217193
vanadate uptake in neurospora crassa occurs via phosphate transport system ii.vanadate, a potent inhibitor of plasma membrane atpases, is taken up by neurospora crassa only when cells are growing in alkaline medium and starving for phosphate. the appearance of a vanadate uptake system (km = 8.2 microm; vmax = 0.15 mmol/min per liter of cell water) occurs under the same conditions required for derepression of a high-affinity phosphate transport system. phosphate is a competitive inhibitor of vanadate uptake, and vanadate is a competitive inhibitor of phosphate uptake. furt ...19836217192
molecular cloning of middle-abundant mrnas from neurospora crassa. 19836197613
folylpolyglutamate synthetase activities of neurospora crassa: nature of products formed by soluble and particulate enzymes in the wild type and polyglutamate-deficient mutants.the folylpolyglutamate synthetase activities of neurospora crassa wild type (fgsc 853) and two polyglutamate-deficient mutants (met-6, 35809, fgsc 1330 and mac, 65108, fgsc 3609) were examined using dialyzed extracts prepared during exponential mycelial growth. enzyme assay was based on incorporation of [u-3h]glutamate in folylpolyglutamates that were separated by gradient elution from deae-cellulose. extracts of the wild type produced h4pteglu2 (15%), h4pteglu3 (35%) and h4pteglu6 (50%) when an ...19836193684
cloning and sequencing of the gene encoding nadp-specific glutamate dehydrogenase in neurospora crassa. 19846233196
isolation and characterization of neurospora mutants affected in invertase synthesis.we have outlined a procedure that allows the large-scale screening of mutagenized neurospora crassa populations for invertaseless mutants. we have isolated and characterized three mutations, inv(dbl1), inv(dbl9) and inv(dbl14), which have been mapped at or near the invertase structural gene. one of these, inv(dbl1), is particularly interesting. our experiments indicate that the reduced level of invertase activity in the inv(dbl1)-containing cell can be explained as the result of a reduced number ...19846232169
induction of cellular efflux by a galactosamine polymer from neurospora crassa.a cationic polymer of d-galactosamine was isolated from culture filtrates of a colonial temperature-sensitive strain of neurospora crassa. adsorption of the polymer to the cell surface initiated immediate efflux of low molecular weight metabolites and subsequent loss of viability. the polymer appeared to bind to those sites on the cell surface that normally bind calcium ions. chemical analysis of the polymer showed it to be partially n-acetylated. the polymer had an isoelectric point of 8.4. thi ...19846233393
calcium inhibits phase shifting of the circadian conidiation rhythm of neurospora crassa by the calcium ionophore a23187.effects of the calcium ionophore, a23187, and antimycin a on the circadian conidiation rhythm of neurospora crassa were examined. a23187 at a concentration of 1 mum in medium not containing divalent cations delayed the phase by 10 hours at ct 10 and advanced it by 5 hours at ct 14 (ct 12 corresponds to the time that discs are transferred from light to dark). this phase shifting was completely inhibited by addition of 0.1 millimolar cacl(2) but not by mgcl(2) at any concentrations examined.antimy ...198416663409
nucleotide sequence of the cloned mrna and gene of the adp/atp carrier from neurospora crassa.a cdna complementary to the mrna of the adp/atp carrier from neurospora crassa was identified among ordered cdna clones by hybridizing total polyadenylated rna to pools of 96 cdna recombinant plasmids and subsequent cell-free translation of hybridization-selected mrna. further carrier cdnas were found by colony filter hybridization at a frequency of 0.2-0.3%. the gene of the carrier was cloned and isolated on a 4.6-kbp ecori fragment of total neurospora dna, and the start of the mrna was determi ...19846325169
molecular analysis of the neurospora qa-1 regulatory region indicates that two interacting genes control qa gene expression.the qa-1 regulatory region controls the expression of the three structural genes required for the early reactions in quinic acid catabolism in neurospora crassa. genetic analysis previously identified two types of noninducible qa-1 mutants, qa-1s and qa-1f, which mapped in separate non-overlapping regions. these mutations were originally interpreted as defining separate domains of a single regulatory protein. this communication describes the further genetic and physical characterization of the q ...19846322189
regulation of a neurospora crassa extracellular rnase by phosphorus, nitrogen, and carbon derepressions.a new extracellular rnase, designated n4, was detected in culture filtrates from neurospora crassa and its regulation was studied. limitation of a nutrient obtainable from rna alone was not sufficient to cause enzyme derepression. the addition of rna to the medium had no inductive effect, but the addition of exogenous protein caused enzyme production. with protein in the medium, n4 was derepressible for all three elemental nutrients obtainable from rna: carbon, nitrogen, and phosphorus. successf ...19846229529
characterization and comparison of a neurospora crassa rnase purified from cultures undergoing each of three different states of derepression.extracellular rnase n4 from neurospora crassa is derepressible by limitation of any of the three nutrient elements obtainable from rna. we have purified and characterized the enzyme from cultures grown under each of the three states of derepression. the purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and gel filtration. we found only one enzyme (n4) that hydrolyzed rna at ph 7.5 in the presence of edta in culture filtrates from nitrogen-, phosphorus-, ...19846229528
a new, rapid and efficient transformation procedure for neurospora.a new, rapid, efficient and reliable method for transforming neurospora crassa is described. in this procedure, germinated conidia are treated with lithium acetate, then incubated with dna, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. optimal conditions to achieve a high transformation rate are reported. transformation can be obtained with both circular and linear plasmid dna and also with genomic dna. although the rate is substan ...198424177533
preliminary characterization of persisting circadian rhythms during space order to evaluate the function of the circadian timing system in space, the circadian rhythm of conidiation of the fungus neurospora crassa was monitored in constant darkness on the sts 9 flight of the space shuttle columbia. during the first 7 days of spaceflight many tubes showed a marked reduction in the apparent amplitude of the conidiation rhythm, and some cultures appeared arrhythmic. there was more variability in the growth rate and circadian rhythms of individual cultures in space tha ...198411539642
assay of rate of aging of conidia of neurospora crassa. 19846233470
transformation of neurospora crassa with the cloned am (glutamate dehydrogenase) gene.we used dna containing the am gene of neurospora crassa, cloned in the lambda replacement vector lambdal-47 (this clone is designated lambdac-10), and plasmid vector subclones of this dna to transform am deletion and point mutant strains. by means of subcloning, all sequences required for transformation to am prototrophy and expression of glutamate dehydrogenase have been shown to reside on a 2.5-kilobase bamhi fragment. we also characterized several am+ strains that were obtained after transfor ...19846230518
glutamate dehydrogenase from wild type neurospora crassa; inactivation by photo-oxidation.the nadp dependent glutamate dehydrogenase from wild type neurospora crassa is inactivated by exposure to light in the presence of the dye, methylene blue. photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. data obtained suggests that the modified group is the same as that reactive to the histidine reagent, diethylpyrocarbonate.19846230273
a chromosome rearrangement in neurospora that produces segmental aneuploid progeny containing only part of the nucleolus translocation t (il leads to vl) oy321 of neurospora crassa a distal portion of the nucleolus organizer chromosome, including ribosomal dna sequences and the nucleolus satellite, is interchanged with a long terminal segment of il. when oy321 is crossed by normal sequence, one-fourth of the meiotic products are segmental aneuploids that contain two copies of the long il segment and that are deficient for the distal portion of the organizer. each such product forms a nucleolus and is viable. th ...19846230215
assay of cytotoxicity and mutagenicity of alkylating agents by using neurospora spheroplasts.a system relying on the use of neurospora crassa spheroplasts has been developed for the assay of cytotoxicity and mutagenicity of chemical compounds. mutagenicity was assayed by using reversion of alleles in the am gene selected to recognize certain specified transitions and also undefined point mutations. cytotoxicity was quantified by measuring a 'cytotoxicity parameter', m, which appears in the exponential function that fits the survival/dose curve for each compound (under standard incubatio ...19846228734
preparation of immobilized nad glycohydrolase from neurospora crassa conidia by hydrophobic interaction--characteristics of the enzyme derivative.nad glycohydrolase from neurospora crassa conidia has been immobilized by hydrophobic interaction on sepharose 4b beads coated with propyl residues through cnbr activation. the bond resulted stable under a wide range of conditions (ionic strength, temperature, ph). as a result of immobilization the ph optimum for catalytic activity shifted by about 0.2 ph unit in the acidic direction, to lie between 7.5 and 7.3. the stability of the enzymatic activity was largely enhanced by effect of immobiliza ...19846096842
organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of neurospora.the organization of the ribosomal dna (rdna) repeat unit in the standard wild-type strain of neurospora crassa, 74-or23-1a, and in 30 other wild-type strains and wild-collected strains of n. crassa, . tetrasperma, n. sitophila, n. intermedia, and n. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic dna, and probing of the southern-blotted dna fragments with specific cloned pieces of the rdna unit from 74-or23-1a. the size of the rdna unit in 74-or23-1a wa ...19846092870
isolation and characterization of a methylammonium resistant mutant of neurospora crassa.a mutant of neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. in contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. however, glutamine still represses these nitrate assimilation enzymes in mea-1. the nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. in addition, mea-1; nit-2 double mutants cannot ut ...198424177912
conidia induce the formation of protoperithecia in neurospora crassa: further characterization of white collar mutants.the treatment of undifferentiated mycelia with heavy suspensions of their own conidia triggers protoperithecial development. this effect was also observed with white collar (wc) mutants and suggests that the wc genes are not structural genes necessary for morphogenesis of protoperithecia but that they are probably involved in regulation.19846235212
isolation of new white collar mutants of neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia.white collar (wc) mutants of neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. eight new wc mutants have been isolated after uv mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. all of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. it is shown that the wc mutations impair another blue light effe ...19846235211
participation of an extracellular deaminase in amino acid utilization by neurospora crassa.a strain of neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. production of the deaminase is subject to ammonium repression. the relationship between the ability of an amino acid to trigger deaminase ...19846235210
major extracellular protease of neurospora crassa.the inducible extracellular alkaline protease of neurospora crassa was demonstrated to be a glycoprotein containing d-galactose residues by use of the enzyme-lectin conjugate horseradish peroxidase-ricinus communis-agglutinin-120. the carbohydrate moiety of the protease appears to be a poor antigen since an antiserum made to the native enzyme recognizes epitopes determined only by the polypeptide portion of the enzyme. immunochemical techniques were used to quantitatively precipitate protease la ...19846235209
intramolecular recombination as a source of mitochondrial chromosome heteromorphism in neurospora.approximately 20% of the genome is missing and an equivalent amount is under-represented in the mitochondrial chromosome population of a stopper (stp) mutant of neurospora crassa during the stopped phase of its cyclical growth pattern. at this stage, a 21 kb (7.2 mu) circular molecule, one-third the length of the normal chromosome, is the predominant form. a complementary 43 kb (14.6 mu) circle appears upon resumption of growth. the circles arise by reciprocal recombination at or near directly r ...19846088067
extended x-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from neurospora k-edge x-ray absorption spectra have been recorded for the enzyme tyrosinase from neurospora crassa, in its oxy, resting (met-aquo), and inhibitor-bound (met-mimosine) forms. the k-edges proper resemble those of oxy- and met-hemocyanin, and confirm the presence of cuii. the forbidden 1s----3d transition is noticeably stronger for the 1-mimosine-bound enzyme, implying some distortion of the tetragonal cu coordination group on inhibitor binding. the extended fine structure (exafs) beyond the k- ...19846234942
mobilization of vacuolar arginine in neurospora crassa. mechanism and role of glutamine.nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of neurospora crassa. the cytosolic arginine is derived from a metabolically inactive vacuolar pool. redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulate ...19846235220
activation of neurospora crassa soluble adenylate cyclase by calmodulin.the soluble form of adenylate cyclase was extracted and purified from wild-type neurospora crassa mycelia. brain or n. crassa calmodulin significantly enhanced this enzyme activity in assay mixtures containing mg2+-atp as substrate. egta reverses this calmodulin activation.19846236798
neurospora circadian rhythms in space: a reexamination of the endogenous-exogenous test the functioning of circadian rhythms removed from periodicities of the earth's 24-hour rotation, the conidiation rhythm of the fungus neurospora crassa was monitored in constant darkness during spaceflight. the free-running period of the rhythm was the same in space as on the earth, but there was a marked reduction in the clarity of the rhythm, and apparent arrhythmicity in some tubes. at the current stage of analysis of our results there is insufficient evidence to determine whether the ...198411540800
glutamine metabolism during aerial mycelium growth of neurospora crassa.during vegetative growth, glutamine is accumulated in the mycelium of neurospora crassa. this high pool of glutamine seems to be required for aerial mycelium growth. enzymes responsible for the synthesis and catabolism of glutamine were measured before and during the partial transformation of a mycelial mat into aerial mycelium. in the transforming mycelial mat,considerable activities of the biosynthetic nadp-glutamate dehydrogenase and glutamine synthetase (predominantly β polypeptide) and also ...198422096812
glutamine requirement for aerial mycelium growth in neurospora crassa.five amino acids are accumulated during vegetative growth of neurospora crassa, particularly.during the prestationary growth phase. alanine, glutamine, glutamate, arginine and ornithine.comprised over 80% of the total amino acid pool in the mycelium. amino acid pools of different amino acid auxotrophs were followed during the partial transformation of a mycelial mat into an aerial mycelium. the mycelial mat under starvation and in direct contact with air rapidly formed aerial mycelium, which pro ...198422096811
production and properties of extracellular endoxylanase from neurospora crassa.neurospora crassa 870 produced 14 and 0.025 u of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; ec and beta-xylosidase (1,4-beta-xylan xylohydrolase; ec per ml, respectively, in 4 days when commercial xylan was used as a carbon source. the effects of ph and carbon sources on xylanase production by n. crassa are discussed. two xylanases (i and ii) were purified and had pi values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. the maximum degree of hydrolys ...198416346591
carbon source regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in neurospora crassa: induction and repression of enzyme synthesis.synthesis and release of nad(p)ase by neurospora crassa wild type was studied in experiments in which mycelia grown in vogel minimal medium were transferred to media containing protein as the only carbon source. several results are presented suggesting that the nad(p)ase may be induced by the presence of protein in the culture medium. low concentrations of sucrose or glucose (0.1%), casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed t ...19846237174
effects of neurospora nuclease halo (nuh) mutants on secretion of two phosphate-repressible alkaline deoxyribonucleases.various recently isolated nuh mutants of neurospora crassa (i.e., mutants which show reduced nuclease haloes on dna-sorbose plates flooded with hcl) were mapped in several new genes or gene clusters and checked for effects on dna repair and nuclease secretion. some of them were found to be sensitive to mms (methylmethane sulfonate) and sterile in meiosis. release of nuclease activities into filtrates of liquid cultures was analyzed by deae-sepharose chromatography. in the wild type, three alkali ...19846235804
the [poky] mutant of neurospora contains a 4-base-pair deletion at the 5' end of the mitochondrial small rrna.[ poky ] and other group i extranuclear mutants of neurospora crassa are characterized by gross deficiencies of mitochondrial small ribosomal subunits and small (19s) rrna. blot-hybridization and other experiments suggest that the 19s rrna (2.0 kilobases) is synthesized via precursors that contain 5'-end extensions. the ratio of precursors to mature rrna is higher in [ poky ] and other group i mutants than in wild type, indicating that the defect involves impaired processing and/or instability o ...19846233613
genetic control of a structural polymer of the neurospora crassa cell wall.the heteropolysaccharide present in fraction 1 of the neurospora crassa cell wall has been characterized in wild-type and morphological mutant strains of this fungus. single and double mutations have been studied to determine possible genetic interactions controlling the chemical composition of such heteropolysaccharides . single mutations studied were peak-2, scumbo ( fgsc 49), ragged ( fgsc 296), and crisp -1 ( fgsc 488). double mutations studied were peak-2, scumbo ( fgsc 419), and ragged cri ...19846233265
cell-biology of ageing. iii. malondialdehyde as an index of free radical reactions in the early senescent mutants of neurospora crassa and study of the effect of free radical scavengers on malondialdehyde contents.malondialdehyde - a product of lipid peroxidation due to free radical reaction was estimated in the culture filtrates of early senescent mutants of neurospora crassa and the effects of vitamin e, vitamin c and sodium selenite (free radical scavengers) in malondialdehyde contents were studied. from the results obtained, it could not be established that increased free radical reaction was the sole factor for the early senescence of all the mutants; and the free radical scavengers had very little e ...19846234997
polyphosphate-cation interaction in the amino acid-containing vacuole of neurospora crassa.the vacuoles of neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-p. vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. polyphosphate was found to be the only macromolecular polyani ...19846232273
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