disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex i in neurospora crassa by repeat induced point mutation (rip).we have used the procedure of sheltered rip to generate mutants of the 78-kda protein of the peripheral arm of neurospora crassa complex i. the nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. this demonstrates that the gene does not serve an essential function. sequence analysis of one allele shows that 61 transition mutations were created ...19957614557
the fungal h(+)-atpase from neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal.fusicoccin affects several physiological processes regulated by the plasma membrane h(+)-atpase in higher plants while other organisms having p-type h(+)-atpases (e.g., fungi) are fusicoccin-insensitive. we have previously shown that fusicoccin binding to its receptor is necessary for h(+)-atpase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the h(+)-atpase from maize. in this paper we show that fusicoccin sensitivity can be conferred on t ...19957878025
folylpolyglutamate synthesis in neurospora crassa: transformation of polyglutamate-deficient mutants.the methionine auxotrophy of neurospora crassa met-6 and mac mutants is related to an inability to synthesize long-chained folylpolyglutamates. both of these lesions affect folylpolyglutamate synthetase activity, but it is not clear whether these mutations occur in different genes or in functional domains of the same gene. to address this question, copies of the met-6+ gene have been introduced into both mutants using plasmid and cosmid vectors. transformation to prototrophy was achieved in both ...19957766162
the vacuolar atpase: sulfite stabilization and the mechanism of nitrate inactivation.using vacuolar membranes from neurospora crassa, we observed that sulfite prevented the loss of vacuolar atpase activity that otherwise occurred during 36 h at room temperature. sulfite neither activated nor changed the kinetic behavior of the enzyme. further, in the presence of sulfite, the vacuolar atpase was not inhibited by nitrate. we tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promot ...19957829484
observation of a secondary tritium isotope effect in the chorismate synthase reaction.chorismate synthase, the seventh enzyme on the shikimate pathway, catalyzes the formation of chorismate from 5-enolpyruvylshikimate 3-phosphate (epsp). this reaction involves the loss of phosphate from c(3) and hydrogen from the c(6) pro-r position of epsp. in order to probe the mechanism of this reaction, [3-(3)h, 14c]epsp has been synthesized and a secondary v/k tritium kinetic isotope measured for the reaction catalyzed by neurospora crassa chorismate synthase. a small but significant value o ...19957819217
purification of deoxyhypusine synthase from neurospora crassa to homogeneity by substrate elution affinity chromatography.deoxyhypusine synthase is an nad(+)-dependent enzyme that catalyzes the formation of deoxyhypusine residue on the eif-5a precursor by using spermidine as the substrate. deoxyhypusine synthase bound tightly to 1,12-diaminododecane-agarose and could be eluted selectively by spermidine. this finding enabled us to develop a simple two-column procedure to purify deoxyhypusine synthase from neurospora crassa to apparent homogeneity. the purified enzyme had a specific activity of 130,000 units/mg of pr ...19957814398
a stringent role of f-actin in the ascogonial differentiation of neurospora crassa fluffy mutant.cytochalasin d, an inhibitor of actin polymerization, interferes with ascogonial differentiation in the fertile fluffy mutant of neurospora crassa. as the total level of actin and its mrna remain unchanged, this suggests that it is in its microfilamentous form (f-form) that actin is stringently required for female differentiation.19958768259
ubiquinol-cytochrome-c oxidoreductase of neurospora crassa. 19958592472
biosynthesis of pteridines in neurospora crassa, phycomyces blakesleeanus and euglena gracilis: detection and characterization of biosynthetic enzymes.occurrence, biosynthesis and some functions of tetrahydrobiopterin (h4biopterin) in animals are well known. the biochemistry of h4biopterin in other organisms than animals was hitherto not widely investigated. recently h4biopterin was found in the phytoflagellate euglena gracilis, in the zygomycete phycomyces blakesleeanus and in the ascomycete neurospora crassa. in euglena, neurospora and phycomyces the enzymatic activities of gtp cyclohydrolase i, 6-pyruvoyl tetrahydropterin synthase and sepia ...19957899493
the hsp70 gene family of neurospora crassa: cloning, sequence analysis, expression, and genetic mapping of the major stress-inducible member.the gene encoding the major heat shock-inducible member of the hsp70 family of neurospora crassa was cloned and characterized. the 5' nontranscribed region shows the presence of consensus sequence motifs resembling the classical heat shock elements found in many heat shock-responsive eukaryotic promoters, as well as metal-responsive-element sequences. the coding region of the gene contains four introns with boundaries and internal consensus motifs typical of genes of filamentous fungi. none of t ...19957798134
isolation of dihydrofolate and folylpolyglutamate synthetase activities from neurospora.the possible association of dihydrofolate synthetase (dhfs) and folylpolyglutamate synthetase (fpgs) in neurospora crassa (fgsc 853, wild type) has been examined using mycelial extracts prepared and fractionated in the presence of protease inhibitors. dhfs and fpgs were assayed by following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. both of these activities were predominately cytosolic in mycelia that were harvested 24 hr ...19957772303
spontaneous mutation during the sexual cycle of neurospora crassa.the dna sequences of 42 spontaneous mutations of the mtr gene in neurospora crassa have been determined. the mutants were selected among sexual spores to represent mutations arising in the sexual cycle. three sexual-cycle-specific mutational classes are described: hotspot mutants, spontaneous repeat-induced point mutation (rips) and mutations occurring during a mutagenic phase of the sexual cycle. together, these three sexual-cycle-specific mutational classes account for 50% of the mutations in ...19957705619
purification and characterization of mitochondrial processing peptidase of neurospora crassa. 19957674958
the effects of temperature change on the circadian clock of neurospora.the phase resetting of the circadian oscillatory system by pulses of increased temperature (zeitgebers) and the temperature compensation of its period length during longer exposures are major features of the system, but are not well understood in molecular terms. in neurospora crassa, the effects of pulses of increased temperature on the circadian rhythm of conidiation were determined and possible inputs to the oscillatory system tested, including changes in cyclic 3',5'-adenosine monophosphate ...19957656690
nuclear actin and rna export in conidial cells of neurospora crassa. 19957542118
genetic and epigenetic inactivation of repetitive sequences in neurospora crassa: rip, dna methylation, and quelling. 19957493491
identification and partial purification of calmodulin-binding microtubule-associated proteins from neurospora crassa.we have purified microtubule-associated proteins from neurospora crassa on the basis of heat stability and affinity to calmodulin. two proteins of molecular masses 170 kda and 190 kda have been partially purified. a third protein of 145 kda was purified almost to homogeneity, and we present evidence that this protein is a specific substrate for a ca2+/calmodulin-dependent protein kinase. the purified 170-, 190-, and 145-kda proteins induce the assembly of microtubules from purified porcine brain ...19948001548
analysis of the dna-binding and dimerization activities of neurospora crassa transcription factor nuc-1.nuc-1, a positive regulatory protein of neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. the carboxy-terminal end of the nuc-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of nuc-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. these upstream sequenc ...19947969122
high frequency repeat-induced point mutation (rip) is not associated with efficient recombination in neurospora.duplicated dna sequences in neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (rip). linked, direct duplications have previously been shown to undergo both rip and deletion at high frequency during premeiosis, suggesting a relationship between rip and homologous recombination. we have investigated the relationship between rip and recombination for an unlinked duplication and for both inverted and direct, linked duplica ...19947896093
heat shock inhibits and activates different protein degradation pathways and proteinase activities in neurospora neurospora crassa, heat shock treatment inhibits proteolytic activity. atp-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. proteinases of 24, 29, and 130 kda were shown to be inhibited by heat shock and were further characterized as to their properties. a major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. during several ...19947813891
cell wall deficiency in "slime" strains of neurospora crassa: osmotic inhibition of cell wall synthesis and beta-d-glucan synthase activity.1. the rcp-3 s/h mutant of neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form of an fz, sg, os-1 ("slime"-like) segregant. the mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (pietro et al. (1990). journal of general microbiology, 136: 121-129). the carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkali-soluble galactosaminogl ...19947550004
substrate analogs as mechanistic probes for the bifunctional chorismate synthase from neurospora crassa.analogs of epsp (4-8) have been prepared, and their activity as substrates for the chorismate synthase from neurospora crassa has been characterized kinetically. the enzyme appears to show strict discrimination against substitution at the z-position of the enol ether side chain as well as against substitution at the s-position of the reduced analogs. both the glycolyl and (r)-lactyl analogs 4 and (r)-5 are good substrates, with (r)-5 having a higher v value than the natural substrate. three subs ...19947947820
5-formyltetrahydropteroylpolyglutamates are the major folate derivatives in neurospora crassa conidiospores.5-formyltetrahydropteroylpolyglutamate (5-cho-h4pteglun) is the only reduced folate derivative that is stable to oxidation and alkaline ph. however, no metabolic role has been assigned to this folate derivative, and evidence for its existence in cells has been questioned. recently, serine hydroxymethyltransferase was shown to catalyze the formation of 5-cho-h4pteglun from 5,10-methenyl-h4pteglun (stover, p., and schirch, v. (1990) j. biol. chem. 265, 14227-14233). we have proposed that 5-cho-h4p ...19947961829
polyamine metabolism and growth of neurospora strains lacking cis-acting control sites in the ornithine decarboxylase gene.ornithine decarboxylase (odc) initiates the synthesis of polyamines (putrescine, spermidine, and spermine) and is highly regulated. we wished to know the importance of the control of odc synthesis to the rates of growth and polyamine synthesis in the fungus, neurospora crassa. we identified two control sites of the spe-1 gene, encoding odc. one was an upstream activation region (uar) and the other was the dna encoding the long odc mrna leader, which governs polyamine-mediated repression of enzym ...19947979392
(1,3) beta-glucan synthase activity of neurospora crassa: identification of a substrate-binding protein.(1,3)beta-glucan synthase activity from the filamentous ascomycete neurospora crassa was purified 1300-fold to a specific activity of 14,000 nmol glucose incorporated/min per mg protein. hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. membrane fractions were extracted with tergitol np-40 and a second high-speed particulate fraction was obtained. enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-beta ...19947947947
tertiary conformational changes of the neurospora crassa plasma membrane h(+)-atpase monitored by hydrogen/deuterium exchange kinetics. a fourier transformed infrared spectroscopy approach.attenuated total reflection fourier transform infrared spectroscopy of hydrated films of the neurospora crassa plasma membrane h(+)-atpase has been used to monitor the alpha-helix and beta-sheet contents and amide hydrogen exchange rates of the enzyme in the absence of ligands or locked in several stages of the enzyme catalytic cycle by mgadp, mg-vanadate, and mgatp-vanadate. no difference larger than 2% was found in the alpha-helix or beta-sheet content of the h(+)-atpase in different conformat ...19947961652
complementary dna sequences of the 24 kda and 21 kda subunits of complex i from neurospora.we have cloned and sequenced cdnas coding for two subunits of the peripheral arm of neurospora crassa complex i. the two polypeptides are synthesized as precursor proteins which are processed to mature forms with predicted molecular masses of 24331 and 20982 da.19947947902
a targeted-replacement system for identification of signals for de novo methylation in neurospora crassa.transformation of eukaryotic cells can be used to test potential signals for dna methylation. this approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming dna can influence dna methylation. we developed a robust system to evaluate the potential of dna fragments to function as signals for de novo methylation in neurospora crassa. the requirements of the system were (i) a location in the n. crass ...19947935421
the neurospora transposon tad is sensitive to repeat-induced point mutation (rip).rip (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the neurospora crassa life cycle. nevertheless, an active line-like retrotransposon, tad, was found in a n. crassa strain from adiopodoumé. the possibility was tested that tad might be resistant to rip, or that the adiopodoumé strain might be incompetent for rip. tad elements derived from the adiopodoumé strain were found to be susceptible to rip. in addition, strains lacking active tad elements, in ...19947851763
neurospora mutants affecting polyamine-dependent processes and basic amino acid transport mutants resistant to the polyamine inhibitor, alpha-difluoromethylornithine.polyamines (spermidine and spermine) are required by living cells, but their functions are poorly understood. mutants of neurospora crassa with enhanced or diminished sensitivity to interference with polyamine synthesis, originally selected to study the regulation of the pathway, were found to have unexpected defects. a group of four non-allelic mutations, causing no interference with polyamine synthesis, each imparted spermidine auxotrophy to a genotype already partially impaired in spermidine ...19947851762
endosomal accumulation of ph indicator dyes delivered as acetoxymethyl esters.intracellular distributions of the putative cytosolic ph indicator dyes bcecf [2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein], c.snarf [5(and 6)-carboxy-seminaphthorhodafluor-1], and c.snarf-calcein have been examined in neurospora crassa and in murine fibroblasts (nih-3t3 cells) under conditions in which both kinds of cells produce visible microscopic vacuoles. all three dyes were administered in electroneutral forms, with the hydroxyl and carboxyl groups esterified (designated as -am ...19947823037
chromium toxicity in neurospora crassa.a comparative study has been made on the mechanisms of toxicities of trivalent and hexavalent forms of chromium in neurospora crassa. of the two forms, cr6+ is more toxic than cr3+. the toxicity of cr3+ was found to be due to its specific antagonism with iron uptake. fe3+ was found to be very effective in reversing the toxicity of cr3+ by concomitantly suppressing its uptake. that the cr3+ toxicity caused a conditional intracellular iron deficiency was indicated by the decrease in the activities ...19947798896
[cyclic nucleotides and membrane electrogenesis in cell differentiation in neurospora crassa].significance of membrane electrogenic system and cyclic nucleotides for developmental regulation in lower eukaryotes was studied in neurospora crassa. despite of their morphological uniformity, hyphal cells were differentiated by their electrophysiological parameters--membrane potential and input resistance. the cells maintained regulated electric communication through septal pores. such electric communication allowed the apical "cell" to compensate the energy requirements for membrane transport ...20137618293
internal translational initiation in the mrna from the neurospora crassa albino-3 gene.the "ribosome scanning model" for translational initiation predicts that eukaryotic mrnas should, as a rule, be monocistronic. however, cases have recently been described of eukaryotic mrnas producing more than one protein through alternative translational initiation at several different aug codons. the present work reports the occurrence of multiple translational start sites on the mrna of the neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyr ...19947929398
fatty acid biosynthesis in novel ufa mutants of neurospora mutants of neurospora crassa having the ufa phenotype have been isolated. two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. the new mutations map to a different chromosomal location than previously characterized ufa mutations. this implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in neurospora.19948000539
tissue-specific differences of the mitochondrial protein import machinery: in vitro import, processing and degradation of the pre-f1 beta subunit of the atp synthase in spinach leaf and root this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated neurospora crassa f1 beta subunit of atp synthase to the mature size product. temperature optimum for protein import, 20 degrees c, was considerably lower than that found in other systems. in spinach leaf mitochondria, the processing pe ...19947948913
cytoplasmic dynein and actin-related protein arp1 are required for normal nuclear distribution in filamentous fungi.cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a variety of intracellular movements, including minus-end-directed transport of organelles. dynein-mediated vesicle transport is stimulated in vitro by addition of the glued/dynactin complex raising the possibility that these two complexes interact in vivo. we report here that a class of phenotypically identical mutants of the filamentous fungus neurospora crassa are defective ...19947929559
mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa3 and c in neurospora crassa.the cyt-12-12 mutant of neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of n. crassa genomic dna. characterization of the cyt-12+ allele reveals that it encodes the structural gene for cytochrome c. dna sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of ...19947882427
neurospora crassa blue light-inducible gene light induces various physiological, morphological and biochemical reactions in the filamentous fungus neurospora crassa. this light response is accompanied by a global change in gene expression, and several light-inducible transcripts (bli-genes) have been cloned. we isolated the genomic clone of the gene bli-3, whose mrna we have previously shown to be induced 2 minutes after the beginning of illumination. its dna sequence predicted a transcriptional unit of 1050 bp encoding a novel, hydr ...19947866300
the isolation and characterization of a neurospora crassa gene (ubi::crp-6) encoding a ubiquitin-40s ribosomal protein fusion protein.we have isolated and sequenced a neurospora crassa gene encoding a single copy of ubiquitin (ubi) fused to the s27a ribosomal (r) protein. we have opted to name this gene the ubiquitin/cytoplasmic r-protein gene 6 (ubi::crp-6). the ubi::crp-6 gene generates a 700-nucleotide (nt) transcript. it shares a 700-bp regulatory region with the cytoplasmic r-protein gene 5 (crp-5), a gene encoding the n. crassa s26 r-protein. the two genes are transcribed divergently from the common regulatory region and ...19948088539
directed replacement of mt a by mt a-1 effects a mating type switch in neurospora test the functions of a mating type genes, we developed an efficient strategy to select transformants of neurospora crassa in which resident a mating type dna was replaced by cloned dna from the mt a idiomorph. cloned a idiomorphic dna could specify all functions, including fertility, of a mating type, but only when it replaced a dna at the mating type locus. only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. gene replacement events involved the homolog ...19948001795
spontaneous mutation at the mtr locus in neurospora: the molecular spectrum in wild-type and a mutator strain.sequence analysis of 34 mtr mutations has yielded the first molecular spectrum of spontaneous mutants in neurospora crassa. the great majority of the mutations are base substitutions (48%) or deletions (35%). in addition, sequence analysis of the entire mtr region, including the 1472-base pair open reading frame and 1205 base pairs of flanking dna, was performed in both the oak ridge and mauriceville strains of neurospora, which are known to be divergent at the dna level. sixteen sequence differ ...19948001794
cytochalasin b-sensitive actin-mediated nuclear rna export in germinating conidia of neurospora crassa. 19948000364
enzyme inactivation related to a hyperoxidant state during conidiation of neurospora crassa.the conidiation process of neurospora crassa is characterized by three morphogenetic steps: hyphal adhesion, aerial hyphal formation, and production of conidia. previous data indicated the occurrence of a hyperoxidant state at the onset of all three morphogenetic steps. because glutamine synthetase (gs) and the biosynthetic glutamate dehydrogenase [gdh(nadp)] enzymes are susceptible to inactivation by reactive oxygen species, we followed these enzyme activities during conidiation and under diffe ...19947952190
the neurospora crassa chs-2 gene encodes a non-essential chitin synthase.chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development in filamentous fungi. in neurospora crassa, at least three different dna fragments related to chitin synthase have been identified. in this study we cloned, sequenced and characterized the chitin synthase 2 structural gene (designated chs-2). the amino acid sequence deduced from the cloned chs-2 genomic dna fragments is very similar to that of chitin synthase genes is ...19947952169
a high throughput in vitro assay for fungal (1,3)beta-glucan synthase in vitro assay for (1,3)beta-glucan synthase activity from the filamentous ascomycete neurospora crassa, suitable for use as a high throughput screen for enzyme inhibitors is described. samples were added to 25 microliters reaction mixtures in 96-well v-bottom microtiter plates and plates incubated at 22 degrees c. reactions were terminated by the addition of tca and the contents transferred to a milliblot d apparatus containing inotech 201-a glass fiber filters. filters were washed to remove ...19947928688
molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of neurospora the filamentous fungus neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. here we report the characterization of the photoinducible promoter of the albino-3 (al-3) gene, encoding ggpp synthase. we have modified the 5' non-coding sequence of the cloned al-3 gene by deletion and site-directed mutagenesis, and we have tested the residual photoinducibility of the dif ...19947815938
gene inactivation in arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations.chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. we have analysed whether this inactivation can trigger repeat-induced point mutations (rip) as has been observed in neurospora crassa. we have previously characterized transgenic lines of arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were re ...19948058043
large single crystals of the neurospora crassa plasma membrane h+-atpase: an approach to the crystallization of integral membrane proteins.large single crystals of the dodecylmaltoside (ddm) complex of a polytopic integral membrane transport protein, the neurospora plasma membrane h(+)-atpase, have been obtained using an approach that attempts to take into account the possibly radically different physicochemical properties of the protein surfaces and the detergent micellar collar. the overall goal of the crystallization strategy employed was to identify conditions in which the protein surfaces of the ddm-atpase complex are moderate ...199415299430
suppressor mutants of neurospora crassa that tolerate allelic differences at single or at multiple heterokaryon incompatibility loci.allelic differences at any one of at least 11 heterokaryon incompatibility (het) loci in neurospora crassa trigger an incompatibility response: localized cell death at sites of hyphal anastomosis. we have isolated spontaneous and insertional suppressor mutants that are heterokaryon-compatible in spite of allelic differences at one or at several het loci. some intra- and extragenic mutants tolerated allelic differences only at single het loci. multi-tolerant spontaneous mutants were isolated by s ...19948088519
cloning and characterization of the pho-2+ gene encoding a repressible alkaline phosphatase in neurospora crassa.the neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho-2+ was cloned and its nucleotide sequence was determined. an open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. 'activator-independent expression' of ectopically integrated pho-2+ was observed, as noted before for ectopically integrated pho-4+.19948026754
mutants of neurospora crassa that alter gene expression and conidia development.several genes have been identified that are highly expressed during conidiation. inactivation of these genes has no observable phenotypic effect. transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. to identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. mutants were then selected that were resistant to the drug dur ...19948016143
transcriptional activation of a cycloheximide-inducible gene encoding laccase is mediated by cpc-1, the cross-pathway control gene, in neurospora crassa.expression of the laccase gene (lacc) of neurospora crassa is transcriptionally inducible by the protein synthesis inhibitor cycloheximide. a lni-1 mutation, conferring the laccase non-inducible phenotype, was found to be a cpc-1 allele. northern blots probed with plasmid pla1, which carries the lacc gene revealed that the cpc-1 mutation abolishes the induced transcription of the lacc gene, indicating requirement of the cpc-1 gene for transcriptional activation of the lacc gene. in northern blot ...19948208246
molecular evolution. unlocking the secrets of retroviral evolution.studies of a small circular plasmid found in the mitochondria of the fungus neurospora crassa shed unexpected light on the evolution of rna viruses into retroviruses.19947522918
light signals are transduced to the phosphorylation of 15 kda proteins in neurospora crassa.a microsomal fraction prepared from the mycelia of the band (bd) strain of neurospora crassa showed enhanced phosphorylation of two small proteins with molecular masses of around 15 kda (ps15) by the irradiation of the reaction mixture containing [gamma-32p]atp at 0 degrees c for 1 s with blue light (450 nm, 6 mumol/m2/s or 420 nm, 80 mumol/m2/s). the reaction was stopped at 5 s of incubation at 0 degrees c after blue light irradiation. the light effect could not be detected in ps15, when a micr ...19948200450
a simple and efficient system for targeting dna to the am locus of neurospora crassa.we have developed a system of recipient strains and donor plasmids that allows targeting of dna sequences to the am locus of neurospora crassa. a recipient strain was constructed that contains the 3' two-thirds of the bacterial hygromycin b (hy) phosphotransferase-encoding gene (hph) in single copy downstream from the am structural gene encoding nadp-specific glutamate dehydrogenase. plasmids have been constructed that contain am, but with the 5' end of hph downstream from am. resistance to hy c ...19948194755
nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen cladosporium fulvum.the avirulence gene avr9 of the fungal tomato pathogen cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene cf9. the avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. in this paper we present evidence for the induction of avr9 gene expression in c. fulvum grown in vitro under conditions of nitrogen li ...19948190081
amino-acid alterations in the beta-tubulin gene of neurospora crassa that confer resistance to carbendazim and diethofencarb.we have previously shown that increased sensitivity to diethofencarb in the carbendazim(mbc)-resistant f914 strain of neurospora crassa is caused by a single amino-acid change in beta-tubulin, 198glu to gly. three diethofencarb-resistant mutants that are also resistant to mbc were isolated from strain f914. they contained single base-pair-substitution mutations in the beta-tubulin gene. the amino acid changes in beta-tubulin, phe from 250leu, val from 165ala, and ala from 237thr, were responsibl ...19948082187
effect of carbon source and extracellular ph on the acidification of the culture medium and phosphatase excretion in neurospora crassa.exogenous ca2+ at concentrations up to 3.5 mm increases the sucrose-induced acidification of the culture medium when the mold neurospora crassa is grown on low-phosphate (pi) medium at ph 7.8. induction depends on the ph of the culture medium adjusted for conidial inoculation and on the absence of carbon sources generating cytoplasmic acetyl coa. furthermore, the excretion of pi-repressible acid and alkaline phosphatases was not stimulated by increasing exogenous ca2+ levels. we also provide evi ...19948000333
characterization of al-2, the phytoene synthase gene of neurospora crassa. cloning, sequence analysis, and photoregulation.we have cloned the al-2 gene of neurospora crassa and have analyzed its structure and regulation. the gene encodes a 603-residue polypeptide with a segment homologous to prokaryotic and other eukaryotic phytoene synthases. rna measurements showed that the level of al-2 mrna increased over 30-fold in photoinduced mycelia compared with dark-grown mycelia. this observation is consistent with the fact that carotenoid biosynthesis is induced by blue light during growth of n. crassa mycelia. the photo ...19948163509
new mutations that suppress mating-type vegetative incompatibility in neurospora crassa.the mating-type locus in the ascomycete neurospora crassa functions as a vegetative-incompatibility locus during asexual growth such that a+a heterokaryons and a/a partial diploids are inhibited in their growth. in this study, we sought mutations that suppress mating-type associated vegetative incompatibility by using a/a partial diploids. mutants were selected as spontaneous escapes from inhibited growth. suppressors were identified by selecting escapes that retained the capacity to mate with b ...19948200515
the proteolipid subunit of the neurospora crassa vacuolar atpase: isolation of the protein and the vma-3 gene.we have isolated the proteolipid subunit from the vacuolar atpase of neurospora crassa, using ion-exchange chromatography. we have also isolated several cdna clones and the corresponding genomic dna that encodes this subunit. the derived protein sequence indicates that the polypeptide is composed of 161 amino acid residues with an m(r) of 16,328 kda. the gene encoding the proteolipid, named vma-3, is unusual in several respects. it contains four introns and, unlike other fungal genes, has non-co ...19948190074
efficient but aberrant cleavage of mitochondrial precursor proteins by the chloroplast stromal processing peptidase.cytosol-synthesized chloroplast and mitochondrial precursor proteins are proteolytically processed after import by highly specific, metal-dependent soluble enzymes: the stromal processing peptidase (spp) and the matrix processing peptidase (mpp), respectively. we have used in vitro processing assays to compare the reaction specificities of highly purified preparations of pea spp and neurospora crassa mpp, both of which are unable to cleave a variety of 'foreign' proteins. we show that spp can cl ...19948168539
characterization of a membrane fragment of respiratory chain complex i from neurospora crassa. insights on the topology of the ubiquinone-binding site.1. a membrane fragment of complex i from the fungus neurospora crassa was isolated by immunoprecipitation from alkaline-extracted mitochondrial membranes. 2. analysis of the polypeptide composition of this hydrophobic domain of complex i has brought insights on the topology of two subunits of the enzyme, namely the 20.8 and 9.3 kda components. 3. our results indicate that the ubiquinone-binding site of complex i resides in the interface of the peripheral and membrane arms of the enzymes. the sig ...19948013735
correlation of the physical and genetic maps of the centromeric region of the right arm of linkage group iii of neurospora crassa.we have cloned three linked genes serine-1 (ser-1), proline-1 (pro-1) and acetate-2 (ace-2) that lie near the centromere on the right arm of linkage group iii (lgiiir) of neurospora crassa. the ser-1 gene was cloned by sib selection. a chromosomal walk that spans 205 kilobases (kb) was initiated from ser-1. complementation analysis with clones isolated during the walk allowed identification of the pro-1 and ace-2 genes. restriction fragment length polymorphism analysis has confirmed the localiza ...19947912215
negative feedback defining a circadian clock: autoregulation of the clock gene frequency.the frequency (frq) locus of neurospora crassa was originally identified in searches for loci encoding components of the circadian clock. the frq gene is now shown to encode a central component in a molecular feedback loop in which the product of frq negatively regulated its own transcript, which resulted in a daily oscillation in the amount of frq transcript. rhythmic messenger rna expression was essential for overt rhythmicity in the organism and no amount of constitutive expression rescued no ...19948128244
gene inactivation triggered by recognition between dna repeats.this chapter focuses on phenomena of gene inactivation resulting from the presence of repeated gene copies within the genome of plants and fungi, and on their possible relationships to homologous dna-dna interactions. emphasis is given to two related premeiotic processes: methylation induced premeiotically (mip) and repeat-induced point mutation (rip) which take place in the fungi ascobolus immersus and neurospora crassa, respectively. the relationships between these processes and genetic recomb ...19948143804
disruption of the gene coding for the 21.3-kda subunit of the peripheral arm of complex i from neurospora crassa.a 21.3-kda subunit of the peripheral arm of complex i from neurospora is encoded by a single chromosomal gene, nuo-21.3b. it is located on linkage group v of the fungal genome, linked to inl. we have isolated and characterized a genomic clone containing this nuclear gene. a dna fragment containing a portion of the coding region of the gene and upstream flanking sequences was introduced by transformation into a wild-type strain of neurospora crassa. a single copy transformant was selected and cro ...19948126004
a crucial role of the mitochondrial protein import receptor mom19 for the biogenesis of mitochondria.the novel genetic method of "sheltered rip" (repeat induced point mutation) was used to generate a neurospora crassa mutant in which mom19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. deficiency in mom19 resulted in a severe growth defect, but the cells remained viable. the number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. ...19948120088
tad1-1, an active line-like element of neurospora crassa.tad is a line-like retrotransposon of neurospora crassa. the element was originally detected and cloned using the am gene as a transposon trap in hybrid strains derived from a cross of adiopodoume (a wild collected strain) and a laboratory strain devoid of tad elements. we report the cloning and sequencing of an active tad element, tad1-1, which is capable of independent transposition. transposition was demonstrated by screening for transfer of the element from a donor nucleus that contained the ...19947512193
an efficient method for gene disruption in neurospora crassa.the frequency with which transforming dna undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. in such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. a protocol is presented for efficient isolation of neurospora crassa strains carrying a definitive null allele in a target gene. the protocol relies o ...19948121407
sequential gel mobility shift scanning of 5' upstream sequences of the neurospora crassa am (gdh) gene.we have used gel mobility shift assays to scan 1.7 kb of 5' non-coding sequence of the am (glutamate dehydrogenase) gene of neurospora crassa for binding by partially fractionated neurospora proteins. using genetic analysis this region had been shown to play an important role in the control of glutamate dehydrogenase (gdh) expression. gel mobility shift analysis identified three regions to which neurospora proteins bind specifically. two of these corresponded to the two elements previously defin ...19948121395
expression of mitochondrial genes in the germinating conidia of neurospora crassa.the germinating asexual spores (conidia) of neurospora crassa were employed to study steps in the accumulation of transcripts of groups of mitochondrial genes, including those for peptide subunits of cytochrome c oxidase (co), atpase (atp), and apocytochrome b (cob). physically clustered groups of genes were expressed as cohorts: transcripts of the atp8-atp6-mtatp9-co2 genes were almost undetectable in the dormant spores, and they accumulated rapidly as a group immediately after spore activation ...19948289326
dna recognition by the nit2 nitrogen regulatory protein: importance of the number, spacing, and orientation of gata core elements and their flanking sequences upon nit2 binding.nit2, a global positive-acting regulatory protein in neurospora crassa, activates the expression of a series of unlinked structural genes in the nitrogen regulation circuit. nit2 binding sites in the promoter region of the nit-3, alc, and lao genes are very different in sequence context except for the presence of at least two copies of a gata core sequence. changing a single nucleotide of only one of two closely spaced gata core elements abolished nit2 binding, demonstrating their importance for ...19948286388
mutants of chlamydomonas with aberrant responses to sulfur the absence of sulfur, chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (ars). these changes appear to help cells acclimate to a sulfur-deficient environment. the elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. the synthesis of ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables ...199412244220
inactivation of the neurospora crassa gene encoding the mitochondrial protein import receptor mom19 by the technique of "sheltered rip".we have used a technique referred to as "sheltered rip" (repeat induced point mutation) to create mutants of the mom-19 gene of neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. sheltered rip permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. furthermore, the nucleus harboring ...19948138148
characterization of mitochondrial dna topoisomerase i from neurospora crassa.dna topoisomerase i isolated from the lower eukaryote neurospora crassa mitochondria was characterized. molar mass of the enzyme in the native state is 120 kda and 60-65 kda when denatured. the ph optimum of the enzyme is 7.8 and the kcl optimum concentration is 40 mmol/l. this topoisomerase is independent of atp and mg2+. n-ethylmaleimide, 4-chloromercuribenzoate, sds, guanidinium chloride, polyethylene glycol, heparin and ethidium bromide inhibit its activity, while novobiocin, nalidixic acid, ...19947959426
temperature effects on the resetting of the phase of the neurospora circadian rhythm.various temperatures relative to a 25 degrees c control have been applied as phase-resetting agents in release-assay experiments using the conidiation rhythm of the mold neurospora crassa. the larger the difference in temperature from the 25 degrees c control, the stronger the phase-resetting effects. phase-resetting curves of the weak type (type 1) are observed for temperatures up to 28 degrees c and down to 22 degrees c, whereas temperatures above 28 degrees c and less than 22 degrees c genera ...19947949309
characterization of the cit-1 gene from neurospora crassa encoding the mitochondrial form of citrate synthase.we have isolated the cdna and corresponding genomic dna encoding citrate synthase in neurospora crassa. analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. the predicted protein has 469 amino acids and a molecular mass of 52,002 da. the gene is interrupted by four introns. hybridization experiments show that a cit-1 probe binds to two different fragments of genomic dna, which are located on different chromosomes ...19947904043
suppression of gene expression by homologous transgenes.when a wild-type strain of neurospora crassa is transformed with different portions of the carotenogenic albino 1 or albino 3 genes, up to 30-35% of the transformants show an albino phenotype. the albino transformants presented a variety of phenotypes ranging from white or yellow to dark yellow colour. the ectopically integrated sequences provoke a severe impairment of the expression of the endogenous al-1 or al-3 genes. this phenomenon, that has been termed quelling, is found to be spontaneousl ...19947847887
abnormal chromosome behavior in neurospora mutants defective in dna methylation.the function and regulation of dna methylation in eukaryotes remain unclear. genes affecting methylation were identified in the fungus neurospora crassa. a mutation in one gene, dim-2, resulted in the loss of all detectable dna methylation. abnormal segregation of the methylation defects in crosses led to the discovery that the methylation mutants frequently generate strains with extra chromosomes or chromosomal parts. starvation for s-adenosylmethionine, the presumed methyl group donor for dna ...19937505062
effects of osmotic stress and growth stage on cellular ph and polyphosphate metabolism in neurospora crassa as studied by 31p nuclear magnetic resonance spectroscopy.high-resolution 31p-nmr was employed to investigate the effects of growth stage and environmental osmolarity on changes of polyphosphate metabolism and intracellular ph in intact neurospora crassa cells. our study showed that changes of these parameters were growth-dependent. the ratio of polyphosphate to orthophosphate in vacuoles increased from 2.4 to 13.5 in n. crassa as cells grew from early log phase to stationary phase. cytoplasmic ph and vacuolar ph changed, respectively, from 6.91 and 6. ...19938218356
hyperactive recombination in the mitochondrial dna of the natural death nuclear mutant of neurospora neurospora crassa, a recessive mutant allele of a nuclear gene, nd (natural death), causes rapid degeneration of the mitochondrial dna, a process that is manifested phenotypically as an accelerated form of senescence in growing and stationary mycelia. to examine the mechanisms that are involved in the degradation of the mitochondrial chromosome, several mitochondrial dna restriction fragments unique to the natural-death mutant were cloned and characterized through restriction, hybridization, ...19938413272
promoter analysis of the bli-7/eas gene.expression of the neurospora crassa gene bli-7, (identical with eas, and ccg-2), is induced by blue light, as well as glucose- or nitrogen-starvation. a promoter analysis was performed by an assay that does not involve promoter-reporter constructs but rather the insertion of foreign dna into the transcribed sequence. to detect regulatory elements a series of deletions in the upstream region was generated. the inducibility of the gene, in response to the three inducing conditions mentioned, is lo ...19938299154
hypersensitive sites in the 5' promoter region of nit-3, a highly regulated structural gene of neurospora crassa.the nit-3 gene of the filamentous fungus neurospora crassa encodes nitrate reductase, the enzyme which catalyzes the first step in nitrate assimilation. the nit-3 gene is subject to a high degree of regulation by metabolic inducers and repressors, and its expression requires two distinct trans-acting regulatory proteins. hypersensitive sites in the 5' dna sequence upstream of the nit-3 gene were mapped with the use of three different nucleases as molecular probes. six hypersensitive sites, three ...19938226616
structural characterization and expression analysis of the neurospora conidiation gene con-6.the gene con-6 of neurospora crassa is expressed during the formation of asexual spores (conidia), but it is not expressed in mycelium. con-6 mrna appears upon induction of conidiation and reaches high levels at the late stages of conidiation, and in mature conidia. the con6 polypeptide and a con6-beta-gal fusion protein were present at high levels only in free conidia. shortly after spore germination con-6 mrna disappears and the con6 polypeptide is degraded. con6 is a small, hydrophilic polype ...19938224542
formation of morphological differentiation patterns in the ascomycete neurospora crassa.morphological differentiation patterns--among them concentric rings and radial zonations--can be induced in the band-mutant of neurospora crassa by appropriate experimental conditions, in particular by a mere shift of certain salt concentrations in the medium. the role of initial experimental conditions is examined and, furthermore, the influences of artificially induced phase differences are analyzed with respect to pattern formation. while the concentric ring pattern is due to some (endogenous ...19938155572
cytochalasin b-sensitive actin-mediated nuclear rna export in germinating conidia of neurospora crassa. 19938111347
organelle movements in the wild type and wall-less fz;sg;os-1 mutants of neurospora crassa are mediated by cytoplasmic microtubules.the cellular basis of organelle transport in filamentous fungi is still unresolved. here we have studied the intracellular movement of mitochondria and other organelles in the fungus neurospora crassa. four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. organelle movements were visualized by dic optics and computer-enhanced video microscopy. in all cell models the transport of o ...19938282762
regulation of ammonium ion assimilation enzymes in neurospora crassa nit-2 and ms-5 mutant neurospora crassa the nit-2 and nmr-1 (ms-5) loci represent the major control genes encoding regulatory proteins that allow the coordinated expression of various systems involved with the utilization of a secondary nitrogen source. in this paper we examined the effect of the nit-2 and ms-5 (nmr-1 locus) mutations on the regulation of the ammonium assimilation enzymes, glutamine synthetase and glutamate dehydrogenase, which are regulated by the products of these genes; however, glutamate synth ...19937907211
day/night and circadian rhythm control of con gene expression in the filamentous fungus neurospora crassa, several events in the process of conidiation are influenced by light. two genes, con-6 and con-10, which were previously shown to be transcriptionally activated during conidiation and by exposure to light, were found to be unexpressed in mycelium maintained in constant darkness or in constant light. however, when mycelium was shifted from darkness to light, transcripts of both genes appeared and were abundant. upon further illumination both transcript ...19938367490
amplification of the nucleolus organizer region during the sexual phase of neurospora crassa.previously we have shown that the nucleolus organizer region (nor) of neurospora crassa displays frequent size changes during crosses. in these initial studies, we observed that decreases in nor size are far more common than increases. here, we have investigated the inheritance of nor size in a strain with an unusually small nor. we call this strain sno for small nucleolus organizer. we found that progeny that inherit their rdna from sno receive either an nor that is larger than that of sno or, ...19938243164
amino-acid substitutions in the zinc finger of nit2, the nitrogen regulatory protein of neurospora crassa, alter promoter element recognition.nit2, the major nitrogen regulatory protein of neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. the promoter of the structural gene for l-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two nit2 binding sites, each with two copies of a gata core consensus sequence. site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of nit2, which serve ...19938221929
cloning and sequence analysis of the glucoamylase gene of neurospora crassa.a 1.0-kb dna fragment, corresponding to an internal region of the neurospora crassa glucoamylase gene, gla-1, was generated from genomic dna by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known n-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. the fragment was used to screen an n. crassa genomic dna library. one clone contained the gene together with flanking regions and ...19938221928
zero-current potentials in a large membrane channel: a simple theory accounts for complex behavior.flow of ions through large channels is complex because both cations and anions can penetrate and multiple ions can be in the channel at the same time. a modification of the fixed-charge membrane theory of teorell was reported (peng, s., e. blachly-dyson, m. forte, and m. colombini. 1992. biophys. j. 62:123-135) in which the channel is divided into two compartments: a relatively charged cylindrical shell of solution adjacent to the wall of the pore and a relatively neutral central cylinder of sol ...19937694668
recognition of specific nucleotide bases and cooperative dna binding by the trans-acting nitrogen regulatory protein nit2 of neurospora crassa.the nit2 nitrogen regulatory protein of neurospora is a dna binding protein which contains a single cys2/cys2 type finger motif followed immediately by a highly basic region. several different approaches were employed to identify nucleotides which appear to be in contact with nit2 in the dna-protein complex. methylation interference and missing contact analyses with the promoter dna fragment of the l-amino acid oxidase gene showed that all three purines in both of two gata core sequences and the ...19938396761
the 59-kda polypeptide constituent of 8-10-nm cytoplasmic filaments in neurospora crassa is a pyruvate decarboxylase.the fungus neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kda polypeptide (p59nc). we have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (pdc, ec, which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. immunofluorescence analyses of 8-10-nm filaments, as well as ...19938359692
nitrate reductase of neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis.the enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. each polypeptide subunit is approximately 100 kda in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. the heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. two histidine r ...19938355655
the interplay of light and the circadian clock. independent dual regulation of clock-controlled gene ccg-2(eas).ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. we show that in neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutan ...19938278550
physical mapping of meiotic crossover events in a 200-kb region of neurospora crassa linkage group i.we propose a general restriction fragment length polymorphism-based strategy to analyze the distribution of meiotic crossover events throughout specific genetic intervals. we have isolated 64 recombinant chromosomes carrying independent meiotic crossover events in the genetic interval eth-1-un-2 on linkage group i of neurospora crassa. thirty-eight crossover events were physically mapped with reference to a 200-kb region cloned by chromosome walking, using n. crassa lambda and cosmid libraries. ...19938104159
Displaying items 3501 - 3600 of 5889