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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
molecular dosimetry of the chemical mutagen ethyl methanesulfonate. quantitative comparison of the mutagenic potency in neurospora crassa and saccharomyces cerevisiae.extending previous work with e. coli and mammalian cells in culture, forward-mutation frequencies induced by ethyl methanesulfonate (ems) were quantitatively compared in neurospora crassa and saccharomyces cerevisiae under standardized conditions. concomitantly, the actual dose to dna was measured by determining the amount of radioactivity bound to dna after treatment with tritium-labeled ems. after exposure to ems (2.5-50 mm), alkylation levels in n. crassa and s. cerevisiae were similar to tho ...19836218404
anthranilate synthase of neurospora crassa: reaction and labeling with glutamine analogs. 19826218784
intron within the large rrna gene of n. crassa mitochondria: a long open reading frame and a consensus sequence possibly important in splicing.we describe the sequence of the 2295 nucleotide long intron and 245 nucleotides of the flanking exon sequences within the large (24s) rrna gene of neurospora crassa mitochondria. the intron contains a long open reading frame, which could correspond to ribosomal protein s5. comparison with the corresponding intron of the large rrna gene of yeast mitochondria reveals a single highly homologous 57 nucleotide long sequence, including the sequence (formula; see text), which is present in virtually al ...19826218884
peptide utilization by nitrogen-starved neurospora crassa.peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. growth of a leu-2 mutant of neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a le ...19836219099
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
chromosomal loci of neurospora crassa. 19826219280
cell wall degradation in the autolysis of filamentous fungi.a systematic study on autolysis of the cell walls of fungi has been made on neurospora crassa, botrytis cinerea, polystictus versicolor, aspergillus nidulans, schizophyllum commune, aspergillus niger, and mucor mucedo. during autolysis each fungus produces the necessary lytic enzymes for its autodegradation. from autolyzed cultures of each fungus enzymatic precipitates were obtained. the degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell ...19826219290
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
role of sulfhydryl compounds in the control of tyrosinase activity in neurospora crassa.it is known that neurospora crassa mycelia cultured in standard concentrations (76 to 190 micrograms/ml) of sulfate accumulate a low molecular weight inhibitor of tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; ec 1.14.1.18.1.). this is not observed in cultures grown under sulfate-limiting conditions. the chemical nature of tyrosinase inhibition was investigated. it was shown to be due to the low molecular weight sulfhydryl fraction of the extracts, in which glutathione is ...19826219663
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
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
photoinduction of protoperithecia in neurospora crassa by blue light. 19836220417
transformation of aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of neurospora crassa.relief of an auxotrophic requirement for uridine in aspergillus nidulans strain g191 has been achieved by transformation with a segment of neurospora crassa dna containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. the mitotic stability of such transformants suggests that the dna has integrated into the genome. southern hybridisation analysis of dna isolated from transformants revealed the presence of pbr322 sequences which have integrated into the host genome along ...19836220717
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
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
structure of the trifunctional trp-1 gene from neurospora crassa and its aberrant expression in escherichia coli.the trifunctional trp-1 gene from neurospora crassa was cloned by complementation of a phosphoribosylanthranilate isomerase-deficient mutant of e. coli. a 2.7-kb dna sequence containing trp-1 was determined. homology of the deduced trp-1 polypeptide sequence to the corresponding e. coli proteins is striking; the order of functional domains within trp-1 is nh2-glutamine amidotransferase-indoleglycerolphosphate synthase-phosphoribosylanthranilate isomerase-cooh (nh2-trpg-trpc-trpf-cooh). whereas t ...19836221060
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
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
control of the ornithine cycle in neurospora crassa by the mitochondrial membrane.in neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol. ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline. neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro. nevertheless, when arginine is added to cells a ...19836222031
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
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
uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of neurospora crassa.seven 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
ribosomal rna genes of neurospora crassa: multiple copies and specificities.ribosomal rna genes were isolated from the germinated conidial and mycelial cells of n. crassa by repeated cycles of 3h-dna:rrna reactions followed by hydroxyapatite chromatography. specificity of multiple copies of those rdnas with respect to n. crassa cell types was studied. the fraction of n. crassa germinated conidial in vitro labelled 3h-dna recovered in the presence of rrna isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rdnas obtained in mycelial cel ...19836222241
[microbiological short-time tests for the evaluation of mutagenic potential of chemical substances].during the last 20 years it became much more interesting to test new chemicals as fast as possible for their carcinogenic potency. therefore new test models were developed. mutagenicity seems to be one sign for carcinogenicity. therefore test systems using microorganisms were studied which are influenced by mutagenic substances. these systems are described, first of all the ames-test, using revertants of salmonella typhimurium, secondly the escherichia coli system deficient of dna-polymerase a ( ...19836222262
purification and characterization of an extracellular acid protease from neurospora crassa.an extracellular acid protease was purified 1420-fold from sulfur-starved protein-induced cultures of neurospora crassa. the enzyme was homogeneous as determined by polyacrylamide electrophoresis. the purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and affinity chromatography on sepharose-linked pepstatin. the enzyme is homologous to aspartyl proteases that are characterized by pepstatin inhibition and trypsinogen activation. it is extremely autolytic ...19836222698
evolutionary aspects of accuracy of phenylalanyl-trna synthetase. a comparative study with enzymes from escherichia coli, saccharomyces cerevisiae, neurospora crassa, and turkey liver using phenylalanine analogues.the phenylalanyl-trna synthetases from escherichia coli, saccharomyces cerevisiae, neurospora crassa, and turkey liver activate a number of phenylalanine analogues (tyrosine, leucine, methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid, mimosine, n-benzyl-l- or n-benzyl-d-phenylalanine, and ochratoxin a), as demonstrated by km and kcat of the atp/ppi pyrophosphate exchange. upon complexation with trna, the enzyme-trnaphe complexes show a ...19836222761
a study of the heat-shock response in neurospora crassa.1. neurospora crassa was grown at 28 degrees c for 12 hr and transferred to higher temperatures for 2 hr. 2. cultures labelled with [35s]methionine showed the synthesis of several new proteins in response to heat-shock at 46 to 48 degrees c. 3. major polypeptides of approximate mr 105,000, 99,000, 78,000, 43,000 and 23,000 were detectable in one-dimensional sds-polyacrylamide slab gel electropherograms. 4. 2-d analysis using isoelectric-focussing in the first dimension and electrophoresis in sds ...19836222926
isolation and characterization of light-insensitive mutants of neurospora crassa.as 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
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
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
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
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
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
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
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
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
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 3.2.2.5.) 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
isolation of a bifunctional domain from the pentafunctional arom enzyme complex of neurospora crassa.limited 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
uses of arginaseless cells in the study of polyamine metabolism (neurospora crassa). 19836225934
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
temperature-induced modifications of glycosphingolipids in plasma membranes of neurospora crassa.plasma membranes isolated from a cell-wall-less mutant of neurospora crassa grown at 37 and 15 degrees c display large differences in lipid compositions. a free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees c membranes, while 15 degrees c plasma membranes exhibited a ratio of nearly 2.0. membranes formed under both growth conditions were found to contain glycosphingolipids. cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolip ...19836226315
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
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
kinetic evidence for interacting active sites in the neurospora crassa plasma membrane atpase.the rate of mgatp hydrolysis (v) by the neurospora plasma membrane atpase shows a sigmoid relationship to substrate concentration ( [s] ), which is precisely fit by the equation: v = (vmax x [s]2)/(km + [s]2). this equation describes an enzyme with two substrate-binding sites, both of which must be filled for hydrolysis to occur. at concentrations above 1 mm, both free mg2+ and free atp behave as competitive inhibitors of the atpase. free atp, although not hydrolyzed, can also significantly stim ...19836226663
precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences.many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. this processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. in this report we employ an in vitro system to investigate the role of processing in protein import. intact neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-p ...19836226666
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
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
[isolation and characteristics of the nucleosomes from the mold neurospora crassa]. 19836227467
isolation and characterization of plasma membranes from strains of neurospora crassa with wild type morphology.a variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of neurospora crassa wild type strain 1a. a combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can ...19836227620
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
possible role of a regulatory gene product upon the myo-inositol-1-phosphate synthase production in neurospora crassa.the regulatory effect of inositol on inositol-1-phosphate synthase in neurospora crassa strains was studied. inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22 degrees c. enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. inositol-requiring strains used in our experiments can be divided into two groups. the first ...19836228255
comparison of the vacuolar membrane atpase of neurospora crassa with the mitochondrial and plasma membrane atpases.the vacuolar membrane atpase of neurospora crassa closely resembles the mitochondrial atpase in its substrate specificity, substrate affinity, and sensitivity to the inhibitor n,n'-dicyclohexylcarbodiimide. three different mutants with altered mitochondrial atpase activity, exhibited as 1) resistance to n,n'-dicyclohexylcarbodiimide, 2) enhanced sensitivity to n,n'-dicyclohexylcarbodiimide, and 3) very low specific activity, were found to be unaltered in the vacuolar membrane atpase. the vacuola ...19836228553
biosynthesis and assembly of nuclear-coded mitochondrial membrane proteins in neurospora crassa. 19836228709
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
a colony filter-hybridization procedure for the filamentous fungus neurospora crassa.a colony filter-hybridization procedure for the filamentous fungus neurospora crassa has been developed. the procedure is sensitive enough to detect escherichia coli plasmid pbr322 dna integrated into chromosomal dna in a neurospora transformant. thus, it should facilitate the isolation of nuclear genes by plasmid-rescue procedures.19836229196
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
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
radioassay of the folate-hydrolyzing enzyme activity, and the distribution of the enzyme in biological cells and tissues.a sensitive radioassay method has been developed to quantitate the activity of the folate-hydrolyzing enzyme which catalyzes the hydrolysis of folic acid to pteroic acid and glutamic acid. the method is based on analyzing [2-14c]pteroic acid separated by a thin-layer chromatography on an avicel sf cellulose plate using 0.1 m potassium phosphate buffer, ph 7.0, as a solvent. this method was found to be more sensitive than a conventional photometric method to determine the activity of the folate-h ...19836229614
a chromosome rearrangement in neurospora that produces segmental aneuploid progeny containing only part of the nucleolus organizer.in 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
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
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
isolation and characterization of mms-sensitive mutants of neurospora crassa.seven different mutants that show high sensitivity to mms killing were isolated and mapped at different loci. one group, mms-(sa1), mms-(sa2) and mms-(sa6), showed high sensitivity to mms but not to uv or gamma-rays. another group, mms-(sa4) and mms-(sa5), showed extremely high sensitivity to uv and mms. and mms-(sa3) and mms-(sa7) were moderately sensitive to both uv and mms. mms-(sa4) and mms-(sa1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. ...19846230534
the complete nucleotide sequence of the neurospora crassa am (nadp-specific glutamate dehydrogenase) gene.the complete nucleotide sequence of a 2.7-kb genomic fragment, containing the neurospora crassa am [nadp-specific glutamate dehydrogenase (gdh)] gene, has been determined. the transcription initiation and polyadenylation sites have been defined by s1 mapping. there are at least four initiation sites between 35 and 60 bases downstream of a tataaa sequence. the single polyadenylation site is immediately downstream of a six-nucleotide sequence which is present in the corresponding position in the n ...19836231215
cytochrome b gene of neurospora crassa mitochondria. partial sequence and location of introns at sites different from those in saccharomyces cerevisiae and aspergillus nidulans.we have sequenced a 2614-base pair fragment of the neurospora crassa mitochondrial dna which contains part of the structural gene for apocytochrome b. this gene is split by at least two introns. the sequence reported here begins within one intron, extends through the next exon, another intron 1276 base pairs long, and the last exon which encodes the cooh terminus of cytochrome b. within the 254 amino acids encoded by the two exons, there is a high degree of sequence conservation, 81%, with cytoc ...19846231283
mutation tests in neurospora crassa. a report of the u.s. environmental protection agency gene-tox program.many mutation tests have been developed in neurospora crassa during the almost 40 years of its use in mutation research. these tests detect two major classes of mutation: gene mutation and meiotic nondisjunction. within the first class, forward- and reverse-mutation tests have been used. the forward-mutation tests include those that detect mutations at many loci and at specific loci. both kinds of forward-mutation tests have been done in homokaryons (n) and heterokaryons (n + n'). from the publi ...19846231482
a chicken repetitive dna sequence that is highly sensitive to single-strand specific endonucleases.a dna sequence consisting of the 5-mer agagg repeated tandemly 32 times has been detected in a chicken genomic clone and found to be present in about 2000 copies per chicken genome. this sequence was highly susceptible to single-strand specific endonucleases isolated from aspergillus oryzae (s1) and mung bean, but cleavage by a single-strand specific endonuclease isolated from neurospora crassa occurred only at a ph below 5.5. endonucleolytic cutting of the agagg sequence by the single-strand sp ...19836231528
the relationship of mo, molybdopterin, and the cyanolyzable sulfur in the mo cofactor.reconstitution of the apoprotein of the molybdoenzyme nitrate reductase in extracts of the neurospora crassa mutant nit-1 with molybdenum cofactor released by denaturation of purified molybdoenzymes is efficient in the absence of exogenous moo2-4 under defined conditions. evidence is presented that this molybdate-independent reconstitution is due to transfer of intact mo cofactor, a complex of mo and molybdopterin (mpt), the organic constituent of the cofactor. this complex can be separated from ...19846231887
acidic ribosomal proteins of neurospora crassa.neurospora crassa acidic ribosomal proteins from the high salt-ethanol extract of 80 s ribosomes have been fractionated by deae-cellulose chromatography. six acidic ribosomal proteins were purified. all resemble escherichia coli l7 and l12 in amino acid composition and molecular weight but each has a slightly different net charge at ph 3.2. four have an apparent molecular weight of approx. 14 000, and two have a molecular weight of approx. 14 800. the amino acid compositions and circular dichroi ...19846231958
localization of pyruvate carboxylase in the cells of neurospora crassa.the cell wall of neurospora crassa was digested enzymatically and the cytosolic and the mitochondrial fractions were separated. the activity of pyruvate carboxylase (ec 6.4.1.1) was detected entirely in the cytosolic fraction. this indicates that the location of pyruvate carboxylase of n. crassa is in the cytosol, but is not in the mitochondria; this is different from the situation in animal tissues.19846232147
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
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
cloning and sequencing of the gene encoding nadp-specific glutamate dehydrogenase in neurospora crassa. 19846233196
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
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
selection of conidial longevity mutants of neurospora crassa.conidial survival was measured after incubation in white light at 30 degrees c and 85-100% relative humidity. the heritable median lifespan (mls) of the wild-type ( age0 ) was 22 days. spontaneous short-lived mutants (age-) with mls about 7 days occurred among both sexual and asexual progeny of wild-type at a frequency of about 10%. radiation of conidia with near ultraviolet light increased the mutation frequency about 5-9-fold above spontaneous. the apparent spontaneous reversion frequency was ...20096233461
linkage of conidial longevity determinant genes in neurospora crassa.the longevity of conidia of neurospora crassa was previously defined as their ability to grow after aging in a constant environment. the heritable median lifespan of the wild type is 22 days. heritable mutants with lifespans of 5-7 days were previously selected. the pleiotropic colony phenotype of the mutants greatly facilitates genetical analysis of their inheritance. twenty-eight mutants were mapped by recombinational analysis. all but one were located at genes on one arm of the seven chromoso ...20106233462
dominance and complementation relationships of conidial longevity mutants of neurospora crassa.previously we reported the occurrence of 16 linked conidial longevity determinant genes in neurospora. mutations of those genes are characterized by a reduction of longevity, a pleiotropic morphological defect, and deficiency of five antioxygenic enzymes. on the basis of the linkage and biochemical data, it was proposed that the genes are spatially and functionally redundant. the results of the present investigation support the hypothesis of functional redundancy. all of the mutants examined wer ...20076233463
assay of rate of aging of conidia of neurospora crassa. 19846233470
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
large-scale isolation of the neurospora plasma membrane h+-atpase.a method for the purification of relatively large quantities of the neurospora crassa plasma membrane proton translocating atpase is described. cells of the cell wall-less sl strain of neurospora grown under o2 to increase cell yields are treated with concanavalin a to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. the pellet obtained consists almost solely of concanavalin a-stabilized plasma membrane sheets greatly enriched in ...19846233916
quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of neurospora crassa to yield active nitrate reductase.an assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of neurospora crassa. a number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. for assay, these were mixed with an excess of extract of the nit-1 mutan ...19846234882
activation of nit-1 nitrate reductase by w-formate dehydrogenase.formate dehydrogenase ( fdh ) from clostridium thermoaceticum is a known tungsten enzyme. fdh was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the azotobacter vinelandii uw-45 and neurospora crassa nit-1 reconstitution assays, respectively. tungsten formate dehydrogenase (w- fdh ), containing only a small mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. these results show w- ...19846234890
extended x-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from neurospora crassa.cu 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
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
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
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
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
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
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
accurate transcription of homologous 5s rrna and trna genes and splicing of trna in vitro by soluble extracts of neurospora.we have developed soluble extracts from neurospora crassa capable of accurately and efficiently transcribing homologous 5s rrna and trna genes. the extracts also appear to quantitatively end-process and splice the primary trna transcripts. although the extracts could not transcribe a heterologous (yeast) 5s rrna gene, they did transcribe a yeast trnaleu gene and slowly process the transcripts. in addition, we have developed a novel strategy for rapidly sequencing uniformly labelled rnas using ba ...19846235482
a leucine trna gene adjacent to the qa gene cluster of neurospora crassa.a single trnaleu gene has been localized and sequenced from neurospora crassa. it is located only 375 bp from the qa gene cluster and it is the only trna or 5s rrna gene within this cloned 37 kb region. the gene encodes a trnaleu with the anti-codon aag, and unlike the other nuclear eukaryotic trnaleu (aag) gene sequenced (from c. elegans), contains an intervening sequence of 27 bp. the neurospora trnaleu (aag) is 84% and 73% homologous respectively to the c. elegans and bovine trnaleu (aag), an ...19846235483
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
mutagenicity of neocarzinostatin in neurospora crassa.neocarzinostatin (ncs) is an acidic, single-chain polypeptide of 109 amino acids that has shown some antitumor activity in clinical trials. ncs is mutagenic in reca+ strains of escherichia coli, but not in reca strains; on the other hand, a defect in the nucleotide-excision-repair pathway has no effect on the mutagenicity of ncs in e. coli. similar results are seen in mammalian cells. excision-repair-deficient xeroderma pigmentosum (xp) cells repair ncs-induced dna damage at the same rate as rep ...19846236365
uv-induced recessive lethals in uvs strains of neurospora which are deficient in uv mutagenesis.the frequencies of spontaneous and uv-induced recessive lethal mutations were compared for uv-sensitive and wild-type heterokaryons of neurospora crassa. these heterokaryons were homokaryotic either for one of two alleles of uvs-3, or for uvs-6 or uvs+. for uvs-3, which is known to have mutator effects, spontaneous recessive lethals were found to be 4-6 times more frequent than observed in uvs+. after correction for clonal distribution of spontaneous mutants, an observed 2-fold increase for uvs- ...19846236366
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
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
in vitro reconstitution of nitrate reductase activity of the neurospora crassa mutant nit-1: specific incorporation of molybdopterin.the reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed form a and form b, respectively. application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. in order to demonstrate that the reconstit ...19846237611
a basal unit of valine-sensitive acetolactate synthase of neurospora crassa.valine-sensitivity as well as activity of acetolactate synthase of neurospora crassa was stabilized with 1.2 m potassium phosphate buffer during extraction from mitochondria and early stages of purification, and with 20% glycerol plus 5 mm sodium pyruvate during sephadex g200 gel chromatography. the enzyme was expressed as four molecular species having the molecular weights of about 500,000, 140,000, 68,000 and 51,000, respectively. the first and the third species showed valine-sensitivity, but ...19846237648
processing peptidase of neurospora mitochondria. two-step cleavage of imported atpase subunit 9.subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial f1f0-atpase is a nuclearly coded protein in neurospora crassa. it is synthesized on free cytoplasmic ribosomes as a larger precursor with an nh2-terminal peptide extension. the peptide extension is cleaved off after transport of the protein into the mitochondria. a processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and ...19846237909
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