PMID(sorted descending)
preparation of a cell-free translation system from a wild-type strain of neurospora crassa.we describe the preparation of an in vitro translation system from a wild-type strain of neurospora crassa. the system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. the translation products have minimal background and can be clearly analyzed by sds-polyacrylamide gel electrophoresis. the method of preparation of the lysate is simple, fast and reproducible. the procedure should be readily applicable to other filamentous fungi.19882968852
cloning of methylated transforming dna from neurospora crassa in escherichia arg-2 mutant of neurospora crassa was transformed to prototrophy with a pbr322-n. crassa genomic dna library. repeated attempts to recover the integrated transforming dna or segments thereof by digestion, ligation, and transformation of escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. analyses of a n. crassa transformant demonstrated that the introduced dna was heavily methylated at cytosine residues. this methylation was shown to be responsibl ...19882968501
putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in neurospora crassa.neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. when the pathway was blocked between putrescine and spermidine, orni ...19882968340
ly121019 inhibits neurospora crassa growth and (1-3)-beta-d-glucan synthase. 19882968332
applicability of the equations of freundlich and langmuir to the adsorption of the azo dye procion scarlet on paramorphic colonies of neurospora crassa.experiments on the adsorption of procion scarlet mx-g by normal hyphae and by paramorphic colonies of neurospora crassa were performed at ph 2.5, 4.5 and 6.5 at 30 degrees c. the measured adsorption isotherms were evaluated by the freundlich and langmuir equations. the removal of dye was most effective at ph 2.5 and more dye was adsorbed per unit mass of cells in the paramorphic cultures than in the normal hyphae. the statistical tests showed langmuir's equation to give a better fit to the adsor ...19872968127
an electrophoretic karyotype of neurospora crassa.a molecular karyotype of neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. the migration of all seven n. crassa chromosomal dnas was defined, and five of the seven molecules were separated from one another. the estimated sizes of these molecules, based on their migration relative to schizosaccharomyces pombe chromosomal dna molecules, are 4 to 12.6 megabases. the seven linkage groups were correlated ...19882967910
xanthine dehydrogenase expression in neurospora crassa does not require a functional nit-2 regulatory gene.xanthine dehydrogenase (xdh) is the initial enzyme in the purine catabolic pathway of n. crassa. secondary nitrogen sources such as purines are metabolized when preferred sources of reduced nitrogen (ammonium or glutamine) are unavailable. xdh synthesis is regulated by glutamine repression and uric acid induction. the nit-2 locus is believed to encode a trans-acting positive regulator essential for the expression of genes encoding enzymes involved in secondary pathways of nitrogen acquisition, s ...19882967694
the cross-pathway control gene of neurospora crassa, cpc-1, encodes a protein similar to gcn4 of yeast and the dna-binding domain of the oncogene v-jun-encoded protein.expression of the gene cpc-1 is required for cross-pathway-mediated regulation of amino acid-biosynthetic genes in neurospora crassa. we have cloned cpc-1 and present an analysis of its structure and regulation. the cpc-1-encoded transcript contains three open reading frames, two of which are located in the 720-nucleotide leader segment preceding the cpc-1 coding region. the two leader open reading frames, if translated, would produce peptides 20 and 41 residues in length. the deduced amino acid ...19882967496
lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase a2: evidence from negative-stain electron microscopy using filipin.the channel protein in the mitochondrial outer membrane of neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase a2. when mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. this suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. it is proposed th ...19882967338
plasmid recovery from transformants and the isolation of chromosomal dna segments improving plasmid replication in neurospora crassa.the efficient recovery of plasmid dna from neurospora crassa transformants is described. lithium acetate-treated spores were transformed with plasmid dna and grown in mass in liquid culture. the resulting mycelial growth was harvested and plasmid dna was extracted and used to transform e. coli to ampicillin resistance. although at low frequency, routine recovery of plasmid psd3 which carries the neurospora qa-2+ gene and pbr322 sequences has been demonstrated. about 10% of the recovered plasmids ...19852967124
regulation of synthesis and secretion of acid and alkaline phosphatases in neurospora crassa.we show that n. crassa represses the production of acid phosphatase at ph higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic ph. the same profile of acid phosphatase production was observed in the pho-2a, pho-3a, nuc-1a, nuc-2a and pregc mutant strains. we also show that acid phosphatase synthesized by the pregc mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by t ...19872967123
mitochondrial protein import: identification of processing peptidase and of pep, a processing enhancing protein.transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. we have isolated the processing activity from neurospora crassa. the final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (mpp, 57 kd) and a processing enhancing protein (pep, 52 kd). the two components were isolated as monomers. pep is about 15-fold more abundant in ...19882967109
expression of heat shock genes of neurospora crassa: effect of hyperthermia and other stresses on mrna levels.neurospora crassa mycelium was heat shocked for intervals varying from 15-180 min. heat shock mrna was monitored by hybridization of northern blots with the drosophila hsp-70 gene probe and an inducible member of the yeast hsp-70 gene family, yg100. a 2.7 kilobase (kb) transcript, with homology to these two probes, was detected in cultures shocked for 15 min; its levels increased up to 60-90 min and declined thereafter. sodium arsenite, too, induced the synthesis of this transcript. an additiona ...19882967081
the structural gene for a phosphorus-repressible phosphate permease in neurospora crassa can complement a mutation in positive regulatory gene nuc-1.van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. this was unexpected because the nuc-1 host already contained a resident van+ gene. plasmids carrying van+ complemented a nuc-2 mutation as well. probing of rna from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control ...19882966896
ribosomal dna inheritance and recombination in neurospora crassa.the genetic segregation of ribosomal dna (rdna) in neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (nts) sequences of nine laboratory wild-type strains and wild-collected strains. in an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rdna was shown to be inherited in a simple, stable mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rdna types. ...19882966889
transcellular ion currents and extension of neurospora crassa hyphae.hyphae of neurospora crassa, like many other tip-growing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. in order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. omission of k+ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. omission of glucose markedly reduced ...19882966862
on the role of energy metabolism in neurospora circadian clock function.neurospora crassa (bda) mycelia were kept in liquid culture. without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at ct 0-6 hr. pulses of metabolic inhibitors such as vanadate (na3vo4), molybdate (na2moo4 : 2 h2o), n-ethylmaleimide (nem), azide (nan3), cyanide (nacn) and oligomycin phase shift the circadian conidiation rhythm of neurospora crassa. maximal advance pha ...19852966687
effect of the homokaryotic state of the uvs-2 allele in neurospora crassa on formaldehyde-induced killing and ad-3 mutation.formaldehyde was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in neurospora crassa. the test was conducted in 3 two-component heterokaryons (dikaryons) of n. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on formaldehyde-induced killing and the induction of ad-3 mutants. these dikaryons were homokaryotic for uvs-2+ (h-12), homokaryotic for usv-2 (h-59), and heterokaryotic for uvs-2 (h-71). for ...19882966296
damage-resistant dna synthesis in eukaryotes.the molecular basis of sensitivity of ionizing radiation and other damaging agents is not clearly defined in eukaryotes. while a large number of mutants have been described only a few have been demonstrated to have a defect in the repair of damage to dna. an interesting characteristic of a sub-group of these mutants, in different species extending throughout the phylogenetic scale, is the presence of damage-resistant dna synthesis. this phenomenon is observed in cells from individuals with the g ...19882966294
neurospora tryptophan synthase. characterization of the pyridoxal phosphate binding site.tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. tryptophan synthase from neurospora crassa, a homodimer of two 75-kda subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microm. the spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were ...19882966157
amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of neurospora crassa.neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. the nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. the nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. many new nit ...19862965995
neurospora crassa pyruvate dehydrogenase complex: component characterization, catalytic properties and location of translation.we propose a simplified procedure for the purification of the neurospora crassa pyruvate dehydrogenase complex. the purified complex showed four protein bands with apparent mr values of 53,400, 52,900, 49,000 and 36,900 upon sds-polyacrylamide gel electrophoresis. components, e2 and e3, of n. crassa pyruvate dehydrogenase complex were identified, respectively, as polypeptides 49,000 and 53,400. it can be deduced that component e1 is constituted of two subunits with mr values of 52,900 and 36,900 ...19882965602
nitrate assimilation in neurospora crassa: enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures.the nitrate assimilatory pathway in neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. both are alpha 2 type homodimers. enzyme-bound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. one, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. three modes of regulation are imposed on the expression of nitrate assimil ...19882963944
possible link between circadian rhythm and heat shock response in neurospora crassa.3-h pulses of elevated temperatures (30 degrees c, 35 degrees c, 40 degrees c) phase shift the circadian conidiation rhythm of neurospora crassa. the phase and amplitude of the phase response curves (prc) were measured in wild type (frq+) and frequency mutants (frq 1, frq 7). the dose dependence of the phase shifts was compared to the dose dependence of total protein synthesis inhibition and heat shock protein induction in the three strains. all processes showed an almost linear dependence on te ...19872963703
on the role of protein synthesis in the circadian clock of neurospora crassa.inhibitors of protein synthesis reset the biological clocks of many organisms. this has been interpreted to mean either that the synthesis per se of proteins is a step in the oscillatory feedback loop or merely that certain unstable protein(s) are required at certain times of the cycle to complete the feedback loop. we report here that neurospora strains bearing the clock mutation frq-7 are relatively insensitive to the resetting action of the protein-synthesis-inhibitor cycloheximide. protein s ...19882963337
metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of neurospora neurospora crassa, the expression of nit-3, the structural gene which encodes nitrate reductase, is highly regulated and requires both nitrate induction and nitrogen catabolite derepression. the major nitrogen regulatory gene, nit-2, acts in a positive fashion to turn on the expression of nit-3 and other nitrogen-related genes during nitrogen derepression. a second regulatory gene, designated nmr, acts in a negative fashion to repress the expression of nitrate reductase and related enzymes, a ...19882962990
stimulation by mammalian insulin of glycogen metabolism in a wall-less strain of neurospora crassa.addition of bovine insulin to cells of the wall-less variant fgsc4761 of neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) intracellular levels of the glycogen precursor udp-glucose decreased 17-18% (p less than 0.01) within 30 min of insulin addition. 2) cells grown with insulin possessed 40% more glycogen than did control cells. 3) the incorporation of 14c-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nm bovine insulin (p ...19882962852
effects of mammalian insulin on metabolism, growth, and morphology of a wall-less strain of neurospora crassa.addition of mammalian insulin to a nutritionally rich, chemically defined culture medium affects neurospora crassa "slime" (wall-less) cells, as indicated by enhancement of growth, extension of viability at the stationary phase of growth, alteration of morphology, and stimulation of glucose oxidation. bovine, porcine, and recombinant human insulin had similar effects on growth and morphology, while proinsulin, reduced insulin, and several other proteins were inactive. insulin added in the presen ...19882962851
a novel stimulator of protein phosphorylation in neurospora endogenous thermostable activator of protein kinase iii (pkiii) was purified from 100,000 x g supernatants of neurospora crassa mycelial extracts. this 38,000 dalton polypeptide, clearly separable from calmodulin on p-60 gel filtration, specifically stimulated n. crassa pkiii activity on casein or phosvitin "in vitro" phosphorylation. the factor was only present in the initial growth phase of the fungus. the mechanism of pkiii activation and its possible regulatory role are discussed.19872961977
characterization of two allelic forms of neurospora crassa laccase. amino- and carboxyl-terminal processing of a precursor.the complete structures of the laccase genes isolated from two different neurospora crassa wild-type strains are described. the genes were cloned by screening partial genomic dna libraries with a nick-translated laccase-specified 1.36-kilobase sali fragment (germann, u. a., and lerch, k. (1986) proc. natl. acad. sci. u.s.a. 83, 8854-8858) as a hybridization probe. nucleotide sequence analysis revealed the presence of two different allelic forms. they conform to the same structural organization, ...19882961749
regulation of the qa gene cluster of neurospora crassa. 19872961304
isolation and characterization of a neurospora crassa ribosomal protein gene homologous to cyh2 of yeast.we have isolated and characterized a neurospora crassa gene homologous to the yeast cyh2 gene encoding l29, a cycloheximide sensitivity-conferring protein of the cytoplasmic ribosome. the cloned neurospora gene was isolated by cross-hybridization to cyh2. it was sequenced from both cdna and genomic clones. the coding region is interrupted by seven intervening sequences. its deduced amino acid sequence shows 70% homology to that of yeast ribosomal protein l29 and 60% homology to that of mammalian ...19872960953
a eukaryotic repressor protein, the qa-1s gene product of neurospora crassa, is homologous to part of the arom multifunctional enzyme.little is known about the proteins involved in the control of gene expression in eukaryotes. although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. we report here that the qa-1s repressor protein of the fungus neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukary ...19872960822
transcription of neurospora crassa 5 s rrna genes requires a tata box and three internal elements.the sequences required for transcription of neurospora crassa 5s rrna genes have been defined using a comprehensive set of deletion and substitution mutations introduced into two cloned genes. an upstream tata box (consensus tcataga) located at -29 to -24, and three internal regions (d, a and c) localized to +19 to +30, +44 to +57 and +73 to +103, respectively, are absolutely required for transcription in vitro. the tata box fixes the start point of transcription. the a and c regions correspond ...19872960818
secretory protein translocation in a neurospora crassa in vitro system. hydrolysis of a nucleoside triphosphate is required for posttranslational in vitro translocation system has been reconstituted with subcellular fractions from the cell wall-less mutant of neurospora crassa (fz;sg;os-1). prepro alpha factor and invertase, secretory proteins from yeast, were faithfully translocated and glycosylated by neurospora microsomes when presence cotranslationally in the neurospora translation system. when presence cotranslationally in the neurospora translation system, microsomes from canine pancreas(crm) could also translocate and glycosylat ...19872960680
role of siderophores in iron storage in spores of neurospora crassa and aspergillus ochraceus.spores of neurospora crassa 74a are lacking in ferritinlike iron pools, as demonstrated by mössbauer spectroscopic analysis. the cyclic hexapeptide siderophore ferricrocin constituted 47% of the total iron content in spores. after germination and growth, the ferricrocin iron pool disappeared, indicating that the metal was utilized. in spores of aspergillus ochraceus, 74% of the total iron content was bound by ferrichrome-type siderophores. siderophores may function as iron storage forms in funga ...19872960664
mitochondrial porin of neurospora crassa: cdna cloning, in vitro expression and import into mitochondria.cdna encoding porin of neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. the deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. according to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. porin was effic ...19872960519
rearrangement of duplicated dna in specialized cells of neurospora.introduction of dna into neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, neurospora sequences deleted from the host genome, and foreign sequences. the sequences already represented in the host were rearranged at high frequency in a cross. in general, both elements of the duplication, that from the plasmid and that from the ...19872960455
differential synthesis and replication of dna in the neurospora crassa slime mutant versus normal cells: role of carcinogens.small quantities of carcinogens, dl-ethionine, thiotepa, actinomycin d, and 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (ccnu) stimulated in vitro deoxyribonucleic acid (dna) synthesis of the slime mutant of neurospora crassa, while there was practically no effect on the dna from the normal wild type 74a strain. all of these compounds caused increased strand separation in the mutant dna of n. crassa, but no separation of normal dna strands. the growth (in vivo tests) of the n. crassa slime muta ...19872959890
two developmental stages of neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for the heat shock temperature of 45 degrees c, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of neurospora crassa. both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. at the rna level, however, these two developmental stages responded with different kinetics to elevated temperature. heat shock rnas (for hsp30 and hsp83) accumulated and declined more rapidly in spores t ...19872959857
molecular genetic analysis of the pyr-4 gene of neurospora means of s1 nuclease mapping, one transcription origin and three termini are identified for the pyr-4 gene of neurospora crassa, the same origin being used also by escherichia coli on the cloned gene. translation of the clone in mini-cells gives a 50,000 dalton gene product, the same size as that determined for the neurospora native enzyme. putative caat and tata boxes, and upstream and downstream potential secondary structures, are identified.19872959843
orientation of enzymic domains in tryptophan synthase of neurospora crassa: an immunoblot analysis of trp3 mutant products.extracts of 52 trp3 mutants of neurospora crassa were tested for the presence of serologically cross-reacting material by the method of electrophoretic blot analysis. the test antigen was obtained by excision of lightly stained bands of denatured pure tryptophan synthase after sds-polyacrylamide gel electrophoresis. rabbit antisera raised against this antigen neutralized and precipitated native tryptophan synthase. of the 52 strains, 19 exhibited banding patterns similar to wild type on electrop ...19872959839
regulation of carbon and nitrogen flow by glutamate synthase in neurospora crassa.a glycine-resistant neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for nadp-dependent glutamate dehydrogenase (gdh).] this new mutation also conferred resistance to serine and methionine sulphoximine (ms), which are inhibitors of glutamine synthetase (gs). in addition, the mutant obtained grew better on ammonium than the am-132 parental strain. resistance to glycine was not due to increa ...19872959749
isolation and regulation of expression of the neurospora crassa copper metallothionein gene.the n. crassa cumt gene has been cloned and its nucleotide sequence determined. to this end an mt specific undecanucleotide was synthesized and used for cdna synthesis with enriched mt mrna as a template. sequence analysis of the cdna obtained allowed the synthesis of a unique 21mer which was used as a hybridization probe to screen a genomic dna library of n. crassa. several positive clones were isolated and subjected to restriction and sequence analysis. in agreement with the published amino ac ...19872959528
luminescence emission from the cu(i)-thiolate complex in metallothioneins.the luminescence emission properties of cu-metallothioneins (from neurospora crassa, agaricus bisporus and livers of bedlington terriers affected by copper toxicosis) as well as of (cu,zn)-metallothionein from bovine fetal liver are reported. upon excitation in the u.v., these proteins emit a largely red-shifted luminescence with a maximum at 565 nm attributable to the cu(i)-thiolate chromophores of the proteins. differences in the shapes of the spectra and the emission intensity are observed wi ...19872959510
heat shock induces peroxidase activity in neurospora crassa and confers tolerance toward oxidative stress.heat shock treatment of 14-h-old neurospora crassa mycelium, for 1 h at 48 degrees c, led to the induction of high levels of peroxidase (ec. activity. no significant change was observed in the superoxide dismutase content. colonies formed by plating conidial suspensions on sorbose-medium also exhibited high peroxidase activity following exposure to hyperthermia and were found to be resistant to normally toxic doses of h2o2. thus one of the heat shock proteins of n. crassa has the funct ...19872959286
copper accumulation in the cell-wall-deficient slime variant of neurospora crassa. comparison with a wild-type strain.the copper-uptake process in the cell-wall-deficient slime variant of the fungus neurospora crassa was compared with that in a wild-type strain. in both organisms investigated most of the copper is taken up from the culture medium during the exponential growth period. the wild-type strain, however, accumulates much more copper than does the slime variant. the influence of the copper concentration in the culture medium on the amounts of copper accumulated intracellularly suggests separate ways of ...19872959274
a portable signal causing faithful dna methylation de novo in neurospora crassa.methylation of cytosine residues in eukaryotic dna is common, but poorly understood. typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified. neurospora crassa dna containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct dna methylation de novo. dna stripped of its methylation by propagation in escherichia coli was reintroduced into neurospo ...19872958937
carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo.the large subunit of carbamoyl phosphate synthase a [carbon-dioxide: l-glutamine amido-ligase (adp-forming, carbamate-phosphorylating), ec] from neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. we have utilized n. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase a to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase a precursor. we found that carboxy ...19872958846
processing of precursor rnas from mitochondria of neurospora crassa.neurospora mitochondrial dna is transcribed into long molecules containing the information of several genes. processing leads to formation of functionally active rnas. it has been shown previously that when trna sequences are present in these transcripts excision of mrnas occurs at the acceptor stem of these trna sequences. we have investigated the processing of precursor rnas transcribed from a region of the mitochondrial genome devoid of trna genes. this region comprises the genes encoding sub ...20072958778
isolation and characterization of an extracellular lipase from the conidia of neurospora crassa.a triacylglycerol lipase (ec from the conidia of neurospora crassa was purified and characterized. the enzyme was purified by sephadex g-100 column chromatography. homogeneity was checked by page, and isoelectric focusing gave a single band corresponding to a pi of 6.4. the enzyme had an apparent mr 54000 +/- 1000 as determined by gel filtration. sds-page gave a single band of mr 27000, suggesting the presence of two identical subunits. this lipase preferred triglycerides with c16- and ...19872958597
partial characterization of gtp-binding proteins in neurospora.six fractions of gtp-binding proteins separated by gel filtration of a mycelial extract containing membrane components of neurospora crassa were partially characterized. [35s]gtp gamma s bound to gtp-binding protein was assayed by repeated treatments with a norit solution and centrifugation. the binding of [35s]gtp gamma s to gtp-binding proteins was competitively prevented in the presence of 0.1 to 1 mm gtp but not in the presence of atp. these gtp-binding proteins fractionated by the gel colum ...19872956953
three-dimensional structure of nadh: ubiquinone reductase (complex i) from neurospora mitochondria determined by electron microscopy of membrane crystals.nadh: ubiquinone reductase (electron transfer complex i) has been isolated from neurospora crassa mitochondria as a monodisperse protein-phospholipid-triton x-100 complex (1:0.04:0.15, by weight). the enzyme is in the monomeric state, has a protein molecular weight of 610,000 and consists of about 25 different subunits. membrane crystals of the enzyme complex have been prepared by adding mixed phospholipid-triton x-100 micelles and then removing the triton by dialysis. diffraction patterns of th ...19872956429
nucleotide sequence and characterization of the pyrf operon of escherichia coli k12.the pyrf gene of escherichia coli k12, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate (omp) decarboxylase, is part of an operon that includes a downstream gene designated orff. the orff gene product is a small polypeptide of unknown function. the nucleotide sequence of a 1549-base pair chromosomal fragment containing this operon was determined. an open reading frame capable of encoding the 27-kda omp decarboxylase subunit was identified and shown to be the pyrf struc ...19872956254
the influence of copper on the induction of tyrosinase and laccase in neurospora crassa.the influence of copper on the cycloheximide-induced synthesis of the copper-containing enzymes tyrosinase and laccase in neurospora crassa was studied by enzyme activity measurements and immunological means. the amount of active enzyme molecules is far higher when the culture medium is copper-supplemented before cycloheximide induction. the synthesis of the apoproteins is not dependent on the presence of copper. this suggests the existence of a copper-storage protein for which metallothionein i ...19872956123
9(10)-alkoxystearic acids lengthen the circadian period of a fatty acid auxotroph of neurospora crassa.9- and 10-alkoxystearic acids with alkoxy groups of varying size were synthesized by solvomercuration of methyl oleate. these "knobbed" fatty acids were used to supplement a neurospora strain carrying the cel mutation, which confers a partial fatty acid deficiency. supplemental unsaturated or short-chain fatty acids are known to lengthen dramatically the circadian period of spore formation (conidiation) in cel strains. a similar dramatic lengthening was obtained with the alkoxy supplements. the ...20102955422
chorismate synthase: a bifunctional enzyme in neurospora crassa. 20102955201
the arom multifunctional enzyme from neurospora crassa. 19872955200
complementation of area- regulatory gene mutations of aspergillus nidulans by the heterologous regulatory gene nit-2 of neurospora crassa.loss-of-function mutations in the regulatory gene area of aspergillus nidulans prevent the utilization of a wide variety of nitrogen sources. the phenotypes of nit-2 mutants of neurospora crassa suggest that this gene may be analogous to the area gene. transformation has been used to introduce a plasmid containing the nit-2 gene into a. nidulans. the nit-2 gene of neurospora complemented mutations in the area gene, restoring the ability to use a variety of nitrogen sources. this indicated that t ...19872954160
three class i introns in the nd4l/nd5 transcriptional unit of neurospora crassa mitochondria.the overlapping nd4l and nd5 genes of neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. all three intervening sequences are class i introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. they contain long open reading frames (orfs; from 306 to 425 codons long) that are continuous and in frame with th ...19872953954
ornithine decarboxylase from neurospora crassa. purification, characterization, and regulation by inactivation.ornithine decarboxylase, a highly regulated enzyme of the polyamine pathway, was purified 670-fold from mycelia of neurospora crassa that were highly augmented for enzyme activity. the enzyme is significantly different from those reported from three other lower eucaryotic organisms: saccharomyces cerevisiae, physarum polycephalum, and tetrahymena pyriformis. instead, the enzyme closely resembles the enzymes from mammals. the mr = 110,000 enzyme is a dimer of 53,000 da subunits, with a specific a ...19872953728
the neurospora crassa metallothionein gene. regulation of expression and chromosomal location.the promoter region of the neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (münger, k., germann, u. a., and lerch, k. (1985) embo j. 4, 2665-2668). we therefore studied the regulation of expression of the n. crassa metallothionein gene in response to different metal ions (cu2+, cd2+, zn2+, co2+, and ni2+) by northern analysis. only copper led to the induction of metallothionein mrna. in n. crassa cultures inoc ...19872953720
arginine-specific carbamoyl phosphate metabolism in mitochondria of neurospora crassa. channeling and control by arginine.citrulline is synthesized in mitochondria of neurospora crassa from ornithine and carbamoyl phosphate. in mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. we have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. very little of this carbamoyl phosphate esca ...19872953716
purification and characterization of arginase from neurospora crassa.we have purified an enzymatically active form of arginase from a wild-type strain of neurospora crassa to homogeneity. the enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. the enzyme exhibited hyperbolic kinetics at ph 9.5 with an apparent km for arginine of 131 mm. antiserum was prepared against the ...19872953715
differential dna methylation during the vegetative life cycle of neurospora crassa.isotope dilution gas chromatography-mass spectrometry analysis of genomic dnas isolated from the different growth phases of neurospora crassa revealed significant differences in the amounts of 5-methylcytosine; the mol % of 5-methylcytosine was 0.36 in conidia (asexual spores), 0.40 in conidial germlings cultured for 3 h, 0.24 in mycelial cells that had grown exponentially for 6 and 12 h, and 0.40 in stationary-phase mycelial cells. these results indicate an approximate inverse correlation betwe ...19872953709
fungal small nuclear ribonucleoproteins share properties with plant and vertebrate u-snrnps.snrnas with properties closely related to those of the major vertebrate u-snrnas are present in the fungi aspergillus nidulans, neurospora crassa and schizosaccharomyces pombe. these rnas possess a tri-methyl guanosine cap structure and a subset cross-hybridizes with human u1 and u2 clones. in the form of snrnps, snrnas from these fungi as well as from saccharomyces cerevisiae and pea plants are immunoprecipitated by human and anti-sm or anti-(u1)rnp autoimmune antibodies. on micro-injection int ...19872953599
induction and de novo synthesis of uricase, a nitrogen-regulated enzyme in neurospora crassa.two efficient procedures are presented for the purification of the purine catabolic enzyme uricase from neurospora crassa. a specific antiserum for uricase was prepared and used to examine the regulation of uricase expression. even when wild-type cells are growing under full nitrogen repression conditions, they possess a considerable basal level of uricase. induction results in a severalfold increase in the level of this enzyme and reflects de novo enzyme synthesis. identical forms of uricase we ...20072952636
the mitochondrial dna of neurospora crassa: deletion by intramolecular recombination and the expression of mitochondrial genes. 19862952110
expression of qa-1f activator protein: identification of upstream binding sites in the qa gene cluster and localization of the dna-binding domain.the qa-1f regulatory gene of neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. this activator protein was expressed in insect cell culture with a baculovirus expression vector. the activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. one site is located between the divergently transcribed qa-1f and qa-1s regulatory genes, corroborating prior ev ...19872951591
nonsense mutations of the ornithine decarboxylase structural gene of neurospora crassa.ornithine decarboxylase (odc) (ec is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. some have speculated that the enzyme protein may have a role in the synthesis of rrna in addition to its role in catalyzing the decarboxylation of ornithine (g. d. kuehn and v. j. atmar, fed. proc. 41:3078-3083, 1982; d. h. russell, proc. natl. acad. sci. usa 80:1318-1321, 1983). to test this possibility, we sought mu ...19872951589
signal for dna methylation associated with tandem duplication in neurospora crassa.most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of neurospora crassa. the region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5s rrna gene. the repeated elements have diverged about 15% by the occurrence of numerous cg to ta mutations, which probably resulted from deamination of methylated cytosines. most but not all common laboratory strains of n. crassa have methylated duplicated dna at the zeta-eta locus. however, many ...19872951588
metabolic utilization of 57fe-labeled coprogen in neurospora crassa. an in vivo mössbauer study.mössbauer spectra of whole cells of neurospora crassa arg-5 ota aga (a siderophore-free mutant) show that the siderophore coprogen is accumulated inside the cell as an entity. 57fe from 57fe-labeled coprogen is slowly removed from the complex (45% in 27 h). the rate of removal depends on the degree of iron starvation of the cells. the distribution of 55fe from [55fe]coprogen in vacuoles, membranes, and cytoplasm has been also determined. from this it is clear that coprogen is accumulated in the ...19872951253
ascus development in two temperature-sensitive four-spore mutants of neurospora crassa.two nonallelic four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. expression depends on temperature. the four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees c) for fsp-1 or to low temperatures (15-20 degrees c) for fsp-2. heterozygous fsp-1 x fsp-1+ crosses make eight-spored asci at 15-20 degrees c but produce many four- ...19862950989
circadian rhythms in neurospora crassa: membrane composition of a mutant defective in temperature compensation.the cel mutant of neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees c, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. during growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. the lev ...19872950925
purification and characterization of 3-dehydroquinase from escherichia coli.a procedure has been developed for the purification of 3-dehydroquinase from escherichia coli. homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. the subunit mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. the native mr, estimated by gel permeation chromatography on sephacryl s-200 (superfine) and on tsk g3000sw, was in the range 52,000-58,000, indicating that the enzyme is dimeric. the ...19862950851
two n-hydroxylaminopurines are highly mutagenic in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of neurospora crassa.3 purine analogs were tested for their mutagenic activities in the ad-3 forward-mutation test in heterokaryon 12 (h-12) of neurospora crassa. in growing cultures of h-12, the n-hydroxylaminopurines 2-amino-6-n-hydroxylaminopurine (aha) and 6-n-hydroxylaminopurine (hap) are potent and strong mutagens, respectively, whereas 2-aminopurine (ap) is a weak mutagen. aha and hap are about equally mutagenic at low doses, but aha is more mutagenic than hap at high doses. despite their potent mutagenicity ...19872950320
isolation and characterization of a dna-uptake-stimulating protein from the culture medium of neurospora crassa slime strain.a protein fraction was purified to homogeneity from the culture medium of the wall-less (slime) strain of neurospora crassa (fgsc 1118), which proved to be identical with dna-uptake-stimulating factor (designated dusf), which has been described earlier [schablik, m. and szabó, g. (1981) fems microbiol. lett. 10, 395-397]. the quantity of dusf is measured by the amount of [3h]dna uptake by neurospora cells at standard conditions. its relative molecular mass was 230,000. it has an isoelectric poin ...19872949969
sequence analysis and transformation by the catabolic 3-dehydroquinase (qute) gene from aspergillus nidulans.the induction of catabolic 3-dehydroquinase by quinic acid in aspergillus nidulans has been shown to involve transcriptional control and yields a single major 0.8 kb mrna. the nucleotide sequence of the catabolic 3-dehydroquinase qute gene has been determined and contains a single uninterrupted open reading frame of 462 bases encoding a 16,505 da protein of 153 residues. comparison with the corresponding qa2 gene of neurospora crassa reveals the absence of 75 nucleotides encoding 25 amino acids ...19862949740
mitochondrial gene urfn of neurospora crassa codes for a long polypeptide with highly repetitive structure.the mitochondrial dna of neurospora crassa contains a long potential gene, designated urfn, which is located immediately downstream from the co1 gene. these two genes are encoded in different reading frames and overlap by 13 codons. urfn is 633 triplets long and terminates at a uag stop codon. its codon usage is atypical for n. crassa mitochondrial exons and introns, and resembles that of the long open reading frame (orf) of the mitochondrial plasmid present in n. crassa strain mauriceville. mul ...19862949084
a recessive circadian clock mutation at the frq locus of neurospora crassa.a circadian clock mutant of neurospora crassa, the most distinctive characteristic of which is the complete loss of temperature compensation of its period length, maps to the frq locus where seven other clock mutants have previously been mapped. this mutant, designated frq-9, is recessive to the wild-type allele and to each of the other frq mutants; thus, it differs from the other mutants, which show incomplete dominance to wild type and to each other. complementation analysis suggests either th ...19862948874
localization of alkaline phosphatase activity at microbody membranes of neurospora crassa and aspergillus nidulans.hyphal cells of neurospora crassa and aspergillus nidulans, grown in sabouraud glucose broth or in a defined medium with xanthine or its catabolites as the nitrogen source, contained single membrane-bound organelles cytochemically identified as microbodies. modified gomori procedures at the ultrastructural level revealed putative alkaline phosphatase activity sites in thin sections of cells of both species of fungi. microbody membranes displayed electron opaque deposits (lead phosphate) which we ...19862948778
primary structure of the neurospora crassa small subunit ribosomal rna coding region. 19862948156
analysis of mutational lesions of acetate metabolism in neurospora crassa by 13c nuclear magnetic resonance.the adaptation of neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13c nuclear magnetic resonance. extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13c]acetate as the sole carbon source. the label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13c-enriched trehalose was evident, indicating that gluconeoge ...19872947898
involvement of chla, e, m, and n loci in escherichia coli molybdopterin biosynthesis.all molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (mpt). to assist in elucidating the biosynthetic pathway of mpt, two mpt-deficient mutants of escherichia coli k-12 were isolated. they lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a neurospora crassa nit-1 strain, and did not yield form a, a derivative of mpt. by p1 ...19872947896
reversal of a neurospora translocation by crossing over involving displaced rdna, and methylation of the rdna segments that result from translocation oy321 of neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group i. in crosses of translocation x translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to normal. genetic, cytological, and molecular evidence indicates that reversion is the resul ...19862947829
purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of neurospora crassa mitochondria.a simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of neurospora crassa mitochondria is described. after fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. covalent chromatography on thiol-activated sepharose cl-4b allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2- ...20152947635
isolation and partial nucleotide sequence of the laccase gene from neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin.the laccase (benzenediol:oxygen oxidoreductase, ec gene from neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxyl-terminal region of the protein has been determined. the gene was cloned by cdna synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(a) rna isolated from cycloheximide-treated n. crassa cultures as template. based on the nucleotide sequence of the cdna obtained, a unique 21-mer was synthesized and use ...19862947240
cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of neurospora crassa and its use as a dominant selectable marker.we cloned the beta-tubulin gene of neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. the gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. the coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. intron position comparisons between the n. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. the mutatio ...19862946938
organization of the functional domains of anthranilate synthase from neurospora crassa. limited proteolysis studies.treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (ingp) synthase and n-(5'-phosphoribosyl)anthranilate (pra) isomerase activities. sequencing the nh2 terminus of the ingp synthase-pra isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment ...20102946679
compartmental and regulatory mechanisms in the arginine pathways of neurospora crassa and saccharomyces cerevisiae. 19862945985
a study of derepression of nad-specific glutamate dehydrogenase of neurospora crassa.transfer of neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic nad-specific glutamate dehydrogenase (nad-gdh), within 30 min of the shift. immunoprecipitation of in vivo pulse-labelled nad-gdh demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. derepression was shown to be under transcriptional control by using the rna synthesis ...19862944990
genetic characterization of the mutagenic activity of environmental chemicals at specific loci in two-component heterokaryons of neurospora crassa. 19862944123
dnase i hypersensitive sites within the inducible qa gene cluster of neurospora crassa.dnase i hypersensitive regions were mapped within the 17.3-kilobase qa (quinic acid) gene cluster of neurospora crassa. the 5'-flanking regions of the five qa structural genes and the two qa regulatory genes each contain dnase i hypersensitive sites under noninducing conditions and generally exhibit increases in dnase i cleavage upon induction of transcription with quinic acid. the two large intergenic regions of the qa gene cluster appear to be similarly organized with respect to the positions ...19862944110
autogenous regulation of the positive regulatory qa-1f gene in neurospora neurospora crassa, the qa-1f regulatory gene positively controls transcription of all genes in the quinic acid (qa) gene cluster. qa-1f is transcribed at a low, uninduced level but is subject to strong (50-fold), autogenous regulation as well as to control by the negative regulatory gene, qa-1s, and the inducer quinic acid. cloned qa-1f dna sequences hybridize to two related mrnas of 2.9 and 3.0 kilobases. when wild-type (qa-1f+) cultures are transferred to inducing conditions, qa-1f mrna inc ...19852943985
regulation of amino acid synthetic enzymes in neurospora crassa in the presence of high concentrations of amino acids.ornithine carbamoyl transferase and leucine aminotransferase of neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. in the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mm range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. a cpc-1 mutant st ...19862943971
identification of dna repair and damage induced proteins from neurospora crassa.the response of neurospora crassa to dna damage induced by uv irradiation has been studied using two-dimensional polyacrylamide gel electrophoresis (2-d page). whole cell extracts of irradiated and untreated cultures were compared. five polypeptides that show changes in response to dna damage have been identified. several mutagen sensitive strains of neurospora were also tested for polypeptide changes on 2-d page. profiles of whole cell extracts of these mutant strains were compared to wild type ...19862943970
genetic and biochemical identification of the glutamate synthase structural gene in neurospora crassa.neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions. despite the physiological importance of glutamate synthase, little is known about the genetics of its expression. to identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. all mutations are recessive to the wild-type allele and belong to the same complementation group as the previously described en(am)-2 (c24) mutation. two lines of evidence in ...19862943726
molecular recognition of siderophores in fungi: role of iron-surrounding n-acyl residues and the peptide backbone during membrane transport in neurospora crassa.recognition of ferric siderophores in neurospora crassa was found to depend on the number and kind of n-acyl residues that surrounded the iron coordination center. in the coprogen series, uptake decreased in the order of coprogen, neocoprogen i, and neocoprogen ii, indicating that gradual replacement of the n-transanhydromevalonyl groups by n-acetyl groups had an adverse effect on uptake. the reverse effect was observed in the ferrichrome series, where uptake decreased in the order of ferrichrys ...19862943724
regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-d-glucan treatment with detergent and nacl, particulate (1----3)-beta-d-glucan synthase (ec from hansenula anomala or neurospora crassa was dissociated into a "soluble fraction" and a "membrane fraction." each fraction alone was almost inactive, but enzymatic activity could be reconstituted by mixing the two fractions and adding gtp or one of its analogs. based on their lability to heat and to incubation with trypsin, the activity in both fractions is proteinaceous. the active component in t ...19862942941
general method for cloning neurospora crassa nuclear genes by complementation of mutants.we have developed a sib selection procedure for cloning neurospora crassa nuclear genes by complementation of mutants. this procedure takes advantage of a modified n. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of dna with recombinant plasmids containing the n. crassa qa-2+ gene. here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. the identities of the putative c ...19852942762
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