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de novo fatty acid synthesis mediated by acyl-carrier protein in neurospora crassa mitochondria.the acyl-carrier protein (acp) in neurospora crassa mitochondria [brody, s. & mikolajczyk, s. (1988) eur. j. biochem. 173, 353-359] mediated a cerulenin-sensitive, de novo fatty acid synthesis independent of the fatty acid synthetase complex present in the cytoplasm. incubation of mitochondria with [2-14c]malonate labeled only the acp as indicated by autoradiography after sds/page. under these in vitro conditions atp was required for the initial acyl-acp formation, but further elongation require ...19902137086
nucleotide sequence of the gene coding for cyclophilin/peptidyl-prolyl cis-trans isomerase of neurospora crassa. 19902137907
direct evidence for the cytoplasmic location of the nh2- and cooh-terminal ends of the neurospora crassa plasma membrane h+-atpase.reconstituted proteoliposomes containing neurospora plasma membrane h+-atpase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the nh2 and cooh termini of the h+-atpase relative to the lipid bilayer. treatment of the proteoliposomes with trypsin in the presence of the h+-atpase ligands mg2+, atp, and vanadate produces approximately 97-, 95-, and 88-kda truncated forms of the h+-atpase similar to those already known to resu ...19902136741
on the role of ca2(+)-calmodulin-dependent and camp-dependent protein phosphorylation in the circadian rhythm of neurospora crassa.pulses of some ca2+ channel blockers (dantrolene, co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5-9 h) phase shifts of the circadian conidiation rhythm of neurospora crassa. pulses of high ca2+, or of low ca2+, a ca2+ ionophore (a23187) together with ca2+, and other ca2+ channel blockers (la3+, diltiazem), however, caused only minor phase shifts. the effect of these substances (a 23187) and of different temperatures on the ca2+ release from isolated vacuo ...19902159489
mutations that affect circadian rhythms in neurospora crassa can alter the reduction of cytochromes by blue light.we have examined membrane fractions from mutant strains of neurospora crassa that have altered responses to blue light or have altered circadian rhythms. using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. the poky strain was found to have high concentrations of photoreducible cytochrome c. after removal of this cytochrome, however, t ...19902151931
premeiotic instability of repeated sequences in neurospora crassa.maintenance of a steamlined genome is probably important to a free-living fungus. the period between fertilization and karyogamy in the life cycle of neurospora and related fungi provides an ideal time for "genome-cleaning". premeiotic intrachromosomal recombination deletes tandem repeats at high frequency in both homothallic and heterothallic filamentous ascomycetes. this eliminates excess copies of tandemly repeated genes and at the same time favors their homogenization. heterothallic fungi su ...19902150906
circadian rhythms in neurospora crassa: biochemistry and genetics. 19902147375
stearolic acid lengthens the condition period of neurospora crassa carrying the cel mutation. 19902145586
genetic coregulation of longevity and antioxienzymes in neurospora crassa.further analysis of a model of the biochemical genetics of cellular longevity in neurospora crassa confirms and amplifies the hypothesis that antioxienzymes and lifespans are genetically co-regulated. the model consists of seven classes of closely related strains with genetically determined median lifespans ranging from 7 to 90 days and differing by about 15-day intervals. the nuclear gene mutations age- and age+ respectively decrease and increase both lifespans and the constitutive enzyme activ ...19902143165
genes responsive to the alteration of polyamine biosynthesis in neurospora crassa.wild-type neurospora crassa grown in minimal medium was exposed to -difluormethyl ornithine (dfmo), a specific inhibitor of ornithine-decarboxylase (odc-ase) activity. protein-synthesis rates impaired by dfmo were restored by the addition of spermidine. the pattern on sds-acrylamide gels displayed three newly synthesized polypeptides, p27, p31 and p99 after dfmo action in the absence of exogenous polyamine. the odc-ase mutant (spe-1) grown in spermidine-supplemented medium did not show an induce ...19902139806
a kinetic study of the interaction between glycogen and neurospora crassa branching enzyme.the interaction of neurospora crassa branching enzyme (1,4-alpha-d-glucan:1,4-alpha-d-glucan 6-alpha-(1,4-alpha-glucano)-transferase) [ec 2.4.1.18] with substrate glycogen or amylopectin was studied by affinity electrophoresis. by this method, the dissociation constants (k) of the branching enzyme for oyster glycogen (cl-12.2, ocl-6.3) and for potato amylopectin (cl-20, ocl-12.8) were determined to be 13.3 mm and 0.355 mm, respectively. the affinity of the enzyme to the substrate glycogen increa ...19902139655
mode of action of glycogen branching enzyme from neurospora crassa.neurospora crassa branching enzyme [ec 2.4.1.18] acted on potato amylopectin or amylose to convert them to highly branched glycogen-type molecules which consisted of unit chains of six glucose units. the enzyme also acted on the amylopectin beta-limit dextrin, indicating that the enzyme acted on internal glucose chains as well as outer chains. by the combined action of n. crassa glycogen synthase [ec 2.4.1.11] and the branching enzyme, a glycogen-type molecule was formed from udp-glucose. in the ...19902139654
fast induction of translatable mrna by blue light in neurospora crassa wt: the wc-1 and wc-2 mutants are blind.after blue-light irradiation of neurospora crassa (wt) mycelia we observed an increase of about 13 translatable mrna species within a period of 30 min. the induction of translatable mrna species followed a specific temporal pattern which permitted the identification of four distinct classes. one of the translatable mrnas was induced in less than 2 min, while the others showed lag periods of 5, 10 or 20 min from the beginning of illumination. the white collar mutants, wc-1 and wc-2, which do not ...19902138217
preparation of neurospora crassa chromosomes from a cell wall-less strain. 19892532324
secretion of an mr 60000 protein by benomyl-treated cells of neurospora crassa.in the presence of the microtubule inhibitor benomyl at micron concentrations, cells of neurospora crassa wild type strain st. lawrence 74a were found to secrete high amounts of an mr 60 000 protein into the culture medium (about 35 micrograms/ml after a 12 h treatment). the secretion also occurred after treatment with the other antitubulin drugs carbendazim (mbc), nocodazole, thiabendazole, and griseofulvin. this secretion is apparently induced by the specific action of benomyl on n. crassa bet ...19892534075
nuclear gene for mitochondrial leucyl-trna synthetase of neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.we have isolated and characterized the nuclear gene for the mitochondrial leucyl-trna synthetase (leurs) of neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. we have purified mitochondrial leurs protein, determined its n-terminal sequence, and used this sequence information to identify and isolate a full-length genomic dna clone. the 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced ...19892574823
isolation and characterization of a laccase-derepressed mutant of neurospora crassa.laccase from the ascomycete neurospora crassa is an inducible secretory enzyme. production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. the lah-1 mutation is mapped between nit-2 and leu-3 on linkage group i, and it behaved as a recessi ...19892553675
classical and molecular genetic analyses of his-3 mutants of neurospora crassa. ii. southern blot analyses and molecular mechanisms of mutagenicity.previous studies (overton et al., mutation res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3b mutants. in the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. the restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-or23-1a and were ...19892530448
isolation and characterization of the tyrosinase gene from neurospora crassa.a precursor form of neurospora crassa tyrosinase has been identified by western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mrna. the molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. in order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. poly(a) rna isolated from tyrosinase-induced cultures of n. crassa was used as a template for cdna synthes ...19892529259
evidence for an essential histidine residue in the neurospora crassa plasma membrane h+-atpase.the neurospora crassa plasma membrane h+-atpase is rapidly inactivated in the presence of diethyl pyrocarbonate (dep). the reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 m-1.min-1 at ph 6.9 and 25 degrees c. the difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of n-carbethoxyhistidine. no change in the absorbance of the inhibited atpase at 278 nm or in the number of ...19892528992
an ethidium bromide induced mutant of neurospora crassa defective in mitochondrial dna.slow growing mutants of neurospora crassa were obtained by ethidium bromide treatment of the wild type strain. a particular mutant er-3 showed stopper phenotype accompanied by deficient cytochrome spectra. the mutant showed an altered restriction pattern of the mtdna which indicated a deletion of 25,000 bp. the phenotype of the ethidium bromide induced mutant er-3 seem to be related to the loss of several essential genes due to a deletion in its mtdna.19892534062
molecular and classical genetic analyses of his-3 mutants of neurospora crassa. i. tests for allelic complementation and specific revertibility.a collection of 81 his-3 mutants of neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. in these studies, the linearity of the complementation map of the his-3 cistron (webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. in the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment w ...19892529437
a cycloheximide-inducible gene of neurospora crassa belongs to the cytochrome p-450 superfamily. 19892529480
the vacuolar atpase of neurospora crassa contains an f1-like structure.we have explored the structure and subunit composition of the vacuolar atpase of neurospora crassa by investigating the effects of nitrate. inhibition of enzyme activity by nitrate was correlated with dissociation of a complex of peripheral polypeptides from the integral membrane part of the enzyme. surprisingly, this nitrate-induced release of subunits occurred only when nucleotides such as adp, atp, or itp were present. atpase inhibitors that have been proposed to act at the active site preven ...19892527854
calcium activates an electrogenic proton pump in neurospora plasma membrane.calcium ionophoresis into coenocytic cells of neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. this is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi.198916666998
isolation of a gene that down-regulates nitrate assimilation and influences another regulatory gene in the same system.glutamine is the preferred source of nitrogen of neurospora crassa. in its presence and that of the gene product of ms5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. in the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. we report here the isolation, from a cosmid bank, of a gene that complements ms5 and can a ...19892528690
molecular cloning and regulatory analysis of the arylsulfatase structural gene of neurospora crassa.the ars-1+ gene of neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. the ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. cotransformation of an n. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by as ...19892528685
epistasis, photoreactivation and mutagen sensitivity of dna repair mutants upr-1 and mus-26 in neurospora crassa.double mutants were constructed combining mus-26, formerly designated uvs-(sa3b), with other uv-sensitive mutants. tests of sensitivity of these double mutants to uv and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. the uv dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 j/m2. the same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. photoreactivation of uv damage ...19892528064
mutations in nuclear gene cyt-4 of neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial rnas.the nuclear cyt-4 mutants of neurospora crassa have been shown previously to be defective in splicing the group i intron in the mitochondrial large rrna gene and in 3' end synthesis of the mitochondrial large rrna. here, northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial rna processing pathways, including those for cob, coi, coii and atpase 6 mrnas, as well as mitochondrial trnas. defects in these pathways include inhibition of 5' and 3' ...19892478417
ubiquitin expression in neurospora crassa: cloning and sequencing of a polyubiquitin gene.we have cloned and sequenced a polyubiquitin gene from neurospora crassa that is organized in a four repeat-tandem array. the first repeat contains a small intron and the last is fused to an extra glutamine codon. in northern blots, two rna species of 1.3 kb and 0.7 kb hybridize to the isolated clone. the larger ubiquitin (ubi) transcript accumulates after partial inhibition of protein synthesis with cycloheximide, and the smaller one preferentially accumulates in conidia after germination. unex ...19892549509
premeiotic change of nucleolus organizer size in neurospora.we have investigated the heritability of nucleolus organizer region (nor) size in neurospora crassa. by pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" nors and the size of a small interstitial nor. tetrad analysis revealed that changes in nor size occur frequently in the sexual phase. moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. unexpectedly, in ...19892527181
inositol trisphosphate induces calcium release from neurospora crassa vacuoles.inositol 1,4,5-trisphosphate is known to release calcium ions from intracellular stores thought to be parts of endoplasmic reticulum in animal cells. in neurospora crassa, however, inositol 1,4,5-trisphosphate acts on vacuoles stimulating a calcium efflux with a km of 5.28 microm. the calcium release is inhibited effectively by dantrolene. these results were obtained by applying two independent methods, measuring calcium binding to fura-2 and loading vacuoles with 45ca.19892527035
deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of neurospora crassa.deoxyribonucleoside triphosphate (dntp) levels were measured in wild type neurospora and nine mutagen-sensitive mutants, at nine different genes. eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dntps. two patterns were seen. five of the mutants had altered ratios of dntps, with relatively high levels of datp and dgtp and low levels of dctp, but changes in the dttp/dctp ratio did not correlat ...19892527032
studies on the active site of the neurospora crassa plasma membrane h+-atpase with periodate-oxidized nucleotides.the neurospora crassa plasma membrane h+-atpase is inactivated by the periodate-oxidized nucleotides, oatp, oadp, and oamp, with oamp the most effective. inhibition of the atpase is essentially irreversible, because sephadex g-50 column chromatography of the oamp-treated atpase does not result in a reversal of the inhibition. inhibition of the atpase by oamp is protected against by the h+-atpase substrate atp, the product adp, and the competitive inhibitors tnp (2',3'-o-(2,4,6-trinitrocyclohexad ...19892545685
isolation of nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in neurospora crassa.expression of nitrate reductase in neurospora crassa requires the positive action of nit-4, a pathway-specific regulatory gene, which mediates nitrate induction. we report the molecular cloning of the nit-4 gene and present results which suggest that the nit-4 gene is constitutively expressed to yield a low-abundance 2.2-kilobase transcript. these results indicate that the nit-2 major control gene and the nit-4 pathway-specific control gene independently regulate the expression of the nitrate as ...19892567729
effect of the uvs-2 allele of neurospora crassa on the mutagenic potency of two n-hydroxylaminopurines and 2-aminopurine in the ad-3 forward-mutation test.the mutagenic potencies of 3 purine analogs were determined in the ad-3 forward-mutation test in growing cultures of heterokaryon 59 (h-59), a nucleotide excision repair-deficient (uvs-2/uvs-2) 2-component heterokaryon of neurospora crassa. two n-hydroxylaminopurines, 2-amino-6-n-hydroxylaminopurine (aha) and 6-n-hydroxylaminopurine (hap), were potent and strong mutagens, respectively, whereas 2-aminopurine (ap) was a moderate mutagen. dose-response curves showed that aha and hap were about equa ...19892526296
the response time of transcription and translation of the leu-2 gene of neurospora to its inducer, alpha-isopropylmalate, approaches the permissible minimum.the rate of transcription and translation of the leu-2 gene of neurospora crassa was measured after induction by alpha-isopropylmalate. little message of enzyme was found before inducer addition but transcription in the lower eukaryote was found well underway within five minutes after inducer addition, followed in a minute or two by the appearance of functional enzyme. the timing was close to the limit set by rna synthesis and ribosome procession. as a consequence, it seems unlikely that travers ...19892525903
use of transformation to make targeted sequence alterations at the am (gdh) locus of neurospora.specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus of am (nadp-specific glutamate dehydrogenase) of neurospora crassa. two approaches have been successful. one approach used am+-containing vectors capable of integrating at any site in the genome. this technique was used to introduce a specific 700 bp insertion near the am locus and to replace chromosomal sequences near am with plasmid dna. efficiency was low, however, and ma ...19892549376
early response and induced tolerance to cycloheximide in neurospora crassa.incubation of neurospora crassa mycelia with low doses of cycloheximide induces the expression of several genes. after 6 h in the presence of cycloheximide, mycelia become tolerant to further additions of the drug and the rate of protein synthesis exhibits a lower sensitivity to it. the polypeptide pattern is indicative of a stress situation.19892528413
effects of heat shock on the induction of mutations by chemical mutagens in neurospora crassa.preheating of neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 x 10(7) conidia/ml 0.067 m kh2po4-na2hpo4 (ph 7.0) buffer to be treatment at 43 degrees c for 60 min. when protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state ...19892525670
uptake, intracellular binding, and excretion of polyamines during growth of neurospora crassa.in neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. we wished to know what determines these pool sizes. in the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. in a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. however, this was true only after an intracellular "sink," with a c ...19892524999
independent transfer of mitochondrial plasmids in neurospora crassa.in the ascomycete fungus neurospora, the distribution of homologous mitochondrial plasmid dnas in different species and among mitochondrial types of n. crassa suggests that these molecules have moved between lineages of clonally propagated mtdna. here we report direct evidence for independent inheritance of mitochondrial plasmids by sexual reproduction which may help explain the distribution of these molecules among mitochondrial lineages.19892524667
fusion of the mitochondrial outer membrane: use in forming large, two-dimensional crystals of the voltage-dependent, anion-selective channel protein.phospholipase a2 induces crystallization of the channel protein, vdac (also called mitochondrial porin), in the outer membrane of neurospora crassa mitochondria. the channel crystals formed in native membranes typically contain a few hundred unit cells. to increase the size of these membrane crystals for low-contrast electron microscopic imaging and diffraction studies, fusion of the isolated mitochondrial outer membranes was attempted before and after phospholipase treatment. successful fusion ...19892470408
luminescence emission from neurospora copper metallothionein. time-resolved studies.the luminescence lifetime of cu-metallothionein from the fungus neurospora crassa has been studied by the frequency-domain emission technique. lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. binding of hg(ii) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. no quenching by oxygen is found for the hg(ii)-cu-metallothionein adduct. by analogy to model compoun ...19892528343
dna sequence, organization and regulation of the qa gene cluster of neurospora crassa.in neurospora, five structural and two regulatory genes mediate the initial events in quinate/shikimate metabolism as a carbon source. these genes are clustered in an 18 x 10(3) base-pair region as a contiguous array. the qa genes are induced by quinic acid and are coordinately controlled at the transcriptional level by the positive and negative regulators, qa-1f and qa-1s, respectively. the dna sequence of the entire qa gene cluster has been determined and transcripts for each gene have been ma ...19892525625
identification of an arginine carrier in the vacuolar membrane of neurospora crassa.a number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of neurospora crassa. the guanido side chain and l-configuration were found to be important for recognition by the arginine carrier. based upon the specificity of recognition, a reactive arginine derivative (n alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. the latter decomposes ...19892523392
identification and electron microscopic analysis of a chaperonin oligomer from neurospora crassa mitochondria.a 7-fold symmetric particle has been identified in neurospora crassa which is most probably the mitochondrial chaperonin. the particle, about 12 nm in diameter, appears in preparations of cytochrome reductase, and is shown to contain a 60 kd protein which cross-reacts with anti-groel antibodies. results of stem mass measurement suggest that the particle is composed of 14 subunits. a preliminary interpretation of the structure of the particle based on electron microscopy is given. its quaternary ...19892569968
premeiotic disruption of duplicated and triplicated copies of the neurospora crassa am (glutamate dehydrogenase) gene.premeiotic inactivation of duplicated sequences (the rip phenomenon of selker et al.) was studied by tetrad analysis using ectopic copies of am+ (coding for nadp-specific glutamate dehydrogenase) and a missense allele am3, coding for a distinctive form of the enzyme, at the normal locus. in duplication crosses either both gene copies were inactivated or neither. two inactivated am3 derivatives were shown to have undergone methylation and numerous base-pair changes, reflected in losses and gains ...19892529044
bioelectrorheological model of the cell. 2. analysis of creep and its experimental verification.the electrorheological model of the cell proposed in part 1 of this work was used to analyze changes in time of the shape of a cell acted on by a constant-amplitude external alternating electric field, with lossiness of the media taken into account. shear stress in the cell membrane was determined. this model was then subjected to preliminary experimental verification using neurospora crassa (slime) spheroplasts subjected to an external alternating electric field of constant frequency (3 mhz) an ...19892533955
proton nmr studies of a metallothionein from neurospora crassa: sequence-specific assignments by noe measurements in the rotating frame.sequential 1h nmr assignments of a metallothionein from neurospora crassa have been accomplished by the combined use of cosy, 2qf-cosy, hohaha, and rotating-frame noe experiments. all potentially observable resonances were assigned except for the epsilon-nh3 group of the c-terminal lysine. 1h noes, when observed in the laboratory frame and at 500-mhz spectrometer frequency, were negligible in this protein due to the inherent rotational correlation time of the molecule. this difficulty was circum ...19892525920
two kinds of "recombination nodules" in neurospora crassa.two morphological types of recombination nodules, termed early and late, are recognized in neurospora crassa. eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transi ...19892526043
a morphological and genetic analysis of conidiophore development in neurospora crassa.the filamentous fungus neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. these conidia are derived from differentiated aerial structures called conidiophores. the process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (sem) and specific fluorescent probes. the first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeat ...19892524423
change in chromosome number associated with a double deletion in the neurospora crassa mitochondrial chromosome.the mitochondrial genome of neurospora is usually found in a single covalently closed circular 62-kbp dna molecule. we report here that the mitochondrial genome of a phenotypic revertant of a stopper mutant (stp-ruv) is contained primarily in two separate, nonoverlapping, autonomously replicating circular chromosomes. the circles, one about 21 kbp and the other somewhat less than 36 kbp are derived from the most frequent classes of recombinant chromosomes (21 and 41 kbp) in the chromosomal popul ...19892524420
characterization of nitrate reductase deficient mutants of chlorella sorokiniana.after x-ray irradiation, 13 mutants of chlorella sorokiniana incapable of using no(3) (-) as n source were isolated using a pinpoint method. using immunoprecipitation and western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. the latter strains contained different patterns of nitrate reductase partial reactions. all isolates were of the nia-type as indicated by the inducibility of purine hydroxylase i and by complementation of nitrate ...198916666622
isolation of a transposable element from neurospora crassa.a neurospora crassa strain from adiopodoumé, ivory coast, contains multiple copies of a transposable element, tad. the element was detected as a 7-kilobase insertion in two independently isolated spontaneous forward mutants of the am (glutamate dehydrogenase) gene. laboratory strains do not contain tad. all progeny from crosses of the adiopodoumé strain to laboratory strains contain multiple copies. when the element was inserted in am, target sequences of 14 and 17 base pairs were duplicated in ...19892538822
isolation and characterization of cadmium-resistant mutants of neurospora crassa.this study identified and characterized four cadmium-resistant mutants of neurospora crassa. one of these mutants maps to linkage group ii and the other three map to linkage group vii, whereas a naturally occurring resistant trait in a strain from japan resides at a distinct but unmapped locus. transport of cadmium into neurospora cells occurs by more than a single uptake system and involves both energy-dependent and -independent components. the resistant mutants transport cadmium in the same ma ...19892525066
immunological identification of the alternative oxidase of neurospora crassa mitochondria.neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. we used a monoclonal antibody to the alternative oxidase of the higher plant sauromatum guttatum to identify a similar set of related polypeptides (mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of ...19892524649
a small isoform of nadh:ubiquinone oxidoreductase (complex i) without mitochondrially encoded subunits is made in chloramphenicol-treated neurospora crassa.in mitochondria of neurospora crassa grown in the presence of chloramphenicol a small form of nadh:ubiquinone reductase is made in place of the normal electron-transfer-complex i. this smaller enzyme has a molecular mass of approximately 350 kda and consists of (at least) 13 different subunits which are all synthesized in the cytoplasm. the complex i which is normally found in neurospora has a molecular mass of approximately 700 kda and consists of around 30 different subunits, of which at least ...19892523306
13n isotope studies of glutamine assimilation pathways in neurospora crassa.l-[amide-13n]glutamine in neurospora crassa is metabolized to [13n]glutamate by glutamate synthase and to [13n]ammonium by the glutamine transaminase-omega-amidase pathway. the [13n]ammonium released is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. most of the nitrogen is retained during cycling between glutamate and glutamine.19892522094
in vivo control of gluconeogenesis in wild-type neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.the rate of cycloheximide-resistant incorporation of carbon from [14c]alanine and [14c]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. the wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. cycloheximide-resistant 14c-incorporating activity was sensitive to carbon catabol ...19892522093
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. ii. more extensive genetic tests reveal an unexpectedly high frequency of multiple-locus mutations.more extensive genetic tests have been performed on a series of 832 x-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon (h-12) of neurospora crassa, reported earlier (webber and de serres 1965). using new tester strains and techniques for performing large-scale genetic tests (heterokaryon, dikaryon and trikaryon) to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of x-ray-induced ad-3 mutants. these ne ...19892521371
neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.a method is proposed for the purification of the neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on sds-polyacrylamide gel electrophoresis as e3, e1 and e2 with respective mr values of 54,000, 53,000 and 49,000. catalytic properties of the purified co ...19892521564
sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of neurospora crassa.marked differences in the pattern of sedimentation of cellular structures were observed after isopycnic centrifugation of crude cell-free preparations from the neurospora crassa wall-less 'slime' variant and mycelial wild-type strain. kinetic studies of particle sedimentation showed that the various types of subcellular components, as revealed by turbidity, uv absorption, polypeptide patterns, and chitin synthetase activity determinations, sediment independently of one another. an important feat ...19892521563
nad-specific glutamate dehydrogenase of neurospora crassa. cdna cloning and gene expression during derepression.the catabolic nad-specific glutamate dehydrogenase of neurospora crassa is one of the many enzymes regulated by carbon catabolite repression. to achieve an understanding of its regulation, cdna and genomic clones were isolated. total poly(a+) rna from derepressed cells was used for the construction of a cdna library in the expression vector, lambda gt11. by screening this library with a polyclonal antiserum against nad-specific glutamate dehydrogenase, a positive clone with a 0.9-kilobase insert ...19892521336
genes expressed during conidiation in neurospora crassa: characterization of con-8.the filamentous fungus neurospora crassa, by a series of defined changes, differentiates from a mycelium composed of branching hyphae to form dormant spores, called conidia. several genes of unknown function (con genes) that are preferentially expressed during this period have been cloned. transcription of these genes has been examined in conidiation-defective mutants, and the results obtained revealed that con-6, con-8, con-10, con-11 and con-13 are most likely to play a unique role during coni ...19892521382
the biological clock of neurospora in a microgravity environment.the circadian rhythm of conidiation in neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). an experiment was conducted on space shuttle flight sts-9 in order to test this hypothesis; during the first 7-8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscilla ...198911537340
isolation of everted plasma membrane vesicles from neurospora crassa and measurement of transport function. 19892561173
phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in neurospora crassa.neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. we have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist ...19892535268
[antifungal activity of 5-benzilidene pyrrolone and furanone derivatives].the antifungal activity against neurospora crassa of some 5-benzilidene pyrrolone and furanone derivatives was realised. relations between the structure and this biological activity are established with fujita-ban and hansch methods. the preponderant part of lipophilicity, resonance effect and e or z configurations have been showed.19892535109
iron limitation and its effect on membrane proteins and siderophore transport in neurospora crassa.cells of the fungus neurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. three strains were studied: n. crassa 74a (wild type), a siderophore-free mutant n. crassa (arg-5 ota aga) as well as a 'slime' variant of n. crassa which lacks a cell wall. plasma membranes were purified, solubilized and analyzed by one-dimensional sds/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecu ...19892534965
acid phosphatase (ec 3.1.3.2) synthesis by phosphorus regulatory mutant strains of neurospora crassa.1. even though altered forms of acid phosphatase ii were synthesized by the mutant strains nuc-1a and nuc-2a of n. crassa, their synthesis was independent of exogenous phosphate concentrations. 2. synthesis of acid phosphatase i by nuc-2a was also insensitive to exogenous phosphate concentrations. when nuc-1a was grown on a low-phosphate medium, it also produced a heat-labile acid phosphatase in addition to a i-like acid phosphatase. i-like acid phosphatase was not detected in the mycelium of th ...19892531620
duplication of the trna(mmet) and trna(cys) genes and of fragments of a gene encoding a subunit of the nadh dehydrogenase complex in neurospora grassa mitochondrial dna.neurospora crassa mitochondrial dna (mtdna) contains duplications of the trna(mmet) gene upstream of a gene (nd2) encoding a subunit of the nadh dehydrogenase complex and of the trna(cys) gene which is found downstream of the apocytochrome b gene. both duplicated genes are located upstream of the small rrna gene. the duplications are extended to flanking sequences. in the case of the trna(mmet) duplication, two fragments of the nd2 gene are also duplicated. these two fragments, which are not con ...19892525962
deoxyglucose-resistant mutants of neurospora crassa: isolation, mapping, and biochemical characterization.neurospora crassa mutants resistant to 2-deoxyglucose have been isolated, and their mutations have been mapped to four genetic loci. the mutants have the following characteristics: (i) they are resistant to sorbose as well as to 2-deoxyglucose; (ii) they are partially or completely constitutive for glucose transport system ii, glucamylase, and invertase, which are usually repressed during growth on glucose; and (iii) they synthesize an invertase with abnormal thermostability and immunological pr ...19892521617
sensitivity to bleomycin and hydrogen peroxide of dna repair-defective mutants in neurospora crassa.mutations were induced in neurospora which cause increased sensitivity to mms (methyl methane-sulfonate) and other mutagens. genetic analysis of such mus demonstrated that some of them defined new dna repair genes (mus-21, and mus-27 to mus-30), while others represented new alleles in previously known genes. to characterize them further, and especially to identify rec- types which have not yet been found in this species, many mms-sensitive strains were tested for cross-sensitivities to bleomycin ...19892463486
dna methylation and control of genome organization in neurospora crassa. 19882977770
the fluorescein isothiocyanate-binding site of the plasma-membrane h+-atpase of neurospora crassa.the mammalian (na+,k+), ca2+-, and (h+,k+)-atpases contain a well-characterized lysine residue that reacts with fluorescein 5'-isothiocyanate (fitc); enzymatic activity is protected by atp, suggesting that the residue is located in or near the nucleotide-binding domain. in this study, the plasma-membrane h+-atpase of neurospora crassa is also shown to be sensitive to fitc. the reaction occurs with pseudo first-order kinetics, has a pka of 8.0, and is stimulated by mg2+. enzymatic activity is pro ...19882904434
transcellular ionic currents studied by intracellular potential recordings in neurospora crassa hyphae. transfer of energy from proximal to apical cells.membrane potentials, input resistances, and electric coupling in the apical parts of n. crassa growing hyphae were recorded with the aid of intracellular microelectrodes. it was revealed that the apical cells were always depolarized by 10 to 30 mv as compared to the adjacent proximal cells. the septal pore maintained an electrical resistance of 4 to 6 m omega. the calculated values of the endogenous electrical current passing through the septal pore varied between 0.5 and 1 na. electrical isolat ...19882973993
cyclic nucleotide phosphodiesterase activity in neurospora crassa. purification by immunoaffinity chromatography and characterization.monoclonal antibodies to neurospora crassa cyclic nucleotide phosphodiesterase (pde i) were selected by their capacity to inhibit the enzyme activity. the monoclonal immunoglobulin, coupled to sepharose 4b, was used for the affinity purification of pde i activity. after sds-polyacrylamide gel electrophoresis the affinity purified pde i fractions showed a single polypeptide band of about 41 kda. this band reacted in western blots with the above mentioned monoclonal immunoglobulin.19882848723
molecular characterization of the mitochondrial dna of a new stopper mutant er-3 of neurospora crassa.an ethidium bromide-induced stopper mutant of neurospora crassa is characterized at the molecular level. the mutant has two populations of mitochondrial dna: a defective predominant mutant molecule and a basal level of the wild-type molecule. the aberrant dna resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. the deletion endpoints are located in the second intron of the nd5 gene, and in a sequence 250 nucleotides ...19882976009
luminescence from the carbon monoxide derivative of agaricus bispora tyrosinase.the luminescence of the co adduct of two isozymic tyrosinases isolated from agaricus bispora, an edible white mushroom, has been studied. at room temperature the emission appears as a single smooth peak centered at 530 nm with fwhm of 2700 cm-1 and a lifetime of 36 microseconds. the lifetime and wavelength of the emission are virtually unchanged on lowering the temperature from 298 to 77 degrees k. solvent composition affects the wavelength of emission minimally. the emission is quenched by oxyg ...19882975158
polyamine transport in neurospora crassa.polyamine transport in neurospora crassa is concentrative and energy dependent in a dilute buffer. the saturable systems governing the uptake of putrescine (km = 0.6 mm), spermidine (km = ca. 0.24 mm), and spermine (km = 0.07 mm) share components, as indicated by mutual inhibition among the polyamines. in addition, nonsaturable components prevail for putrescine and spermidine, particularly the former. radiolabeled substrates, once in the cell, are released only slowly, even if unlabeled polyamin ...19882975157
metabolism of d-glucose in a wall-less mutant of neurospora crassa examined by 13c and 31p nuclear magnetic resonances: effects of insulin.13c nmr and 31p nmr have been used to investigate the metabolism of glucose by a wall-less strain of neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. with d-[1-13c]- or d-[6-13c]glucose as substrates, the major metabolic products identified from 13c nmr spectra were [2-13c]ethanol, [3-13c]alanine, and c1- and c6-labeled trehalose. several observations suggested the existence of a substantial hexose monophosphat ...19882975509
the heat shock response of neurospora crassa: stress-induced thermotolerance in relation to peroxidase and superoxide dismutase levels.heat shock and other treatments, including cadmium chloride, hydrogen peroxide and sodium arsenite, led to the induction of high levels of peroxidase activity as well as thermotolerance in neurospora crassa. no correlation was apparent between superoxide dismutase levels and development of thermotolerance following exposure to these stress conditions. a prominent role for peroxidase in protection against damage by toxic products of oxygen is suggested.19882847725
the plasma membrane h+-atpase of neurospora crassa. properties of two reactive sulfhydryl groups.previous work with n-ethylmaleimide (nem) has defined two sites on the neurospora plasma membrane h+-atpase. modification of one (the "fast" site) by nem is rapid but does not affect atpase activity, while modification of the other (the "slow" site) inactivates the enzyme and is protectable by mgatp or mgadp. in the present study, a wider array of sulfhydryl reagents have been used to examine the properties of both sites. the results show the following. (a) both fast and slow sites react prefere ...19882903147
translocation of a fragment of invertase across microsomal vesicles isolated from neurospora crassa requires the hydrolysis of a nucleoside triphosphate.the step which requires the hydrolysis of a nucleoside triphosphate for translocation of a protein across microsome was investigated by studying translocation uncoupled from translation using two truncated products of invertase: one product contains the first 262 amino acids of the secreted invertase (inv262); the other, the first 104 amino acids (inv104). the truncated products were translated from rna transcripts without a stop codon. it is demonstrated that the translated products contain an ...19882971655
isolation of genes encoding the neurospora vacuolar atpase. analysis of vma-1 encoding the 67-kda subunit reveals homology to other atpases.the vacuolar membrane of neurospora crassa contains a h+-translocating atpase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. both genomic and cdna clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. the gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, mr 67,121. within the seq ...19882971651
isolation of genes encoding the neurospora vacuolar atpase. analysis of vma-2 encoding the 57-kda polypeptide and comparison to vma-1.in partially purified preparations of the vacuolar atpase from neurospora crassa, the two most prominent components are polypeptides of mr = 70,000 and 60,000. we previously reported the isolation of the gene vma-1, which encodes the mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of atp hydrolysis (bowman, e. j., tenney, k., and bowman, b. j. (1988) j. biol. chem. 263, 13994-14001). we now report the isolation of a gene (designated vma-2), that encodes the ...19882844751
presence of abnormal synaptonemal complexes in heterothallic species of neurospora.synaptonemal complex abnormalities are frequent in reconstructed meiotic prophase nuclei of neurospora crassa and neurospora intermedia. three kinds of synaptonemal complex anomalies were seen: lateral component splits, lateral component junctions, and multiple complexes. the anomalies apparently are formed during or after the pairing process, as they were not seen in the largely unpaired early zygotene chromosomes. their presence at all the other substages from mid-zygotene to late pachytene in ...19882974436
dual roles for calcium ions in apical growth of neurospora crassa.we report initial attempts to define the role of ca2+ in the polarized extension of neurospora crassa. growth of the organism was diminished in media containing less than 1 mm-ca2+; extension was more severely impaired than biomass synthesis, resulting in the formation of stubby, bulbous hyphae, even of spherical cells. reduced extension and abnormal morphology were correlated with the loss of surface-bound ca2+, probably associated with the cell wall. intracellular ca2+ may be represented by ma ...19882978297
binding of a 30-kda protein to the pyruvate kinase gene of neurospora crassa.extracts of a wild-type strain of neurospora crassa, electrophoresed on sds-polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (pk) gene fragment containing the 5' noncoding sequence and a large part of the coding region. a 30-kda protein was found to bind strongly to the pk gene dna, while binding weakly to plasmid puc12 dna and to total n. crassa dna. probing of blots with individual restriction fragments derived from the pk ge ...19882973333
molecular cloning and characterization of a negative-acting nitrogen regulatory gene of neurospora crassa.expression of the structural genes of the nitrogen control circuit of neurospora crassa is regulated by the positive-acting nit-2 control gene and by the negative-acting nmr control gene. nitrate reductase is expressed in a constitutive fashion in nmr mutant strains, which appear to be largely insensitive to nitrogen catabolite repression. thus, nmr mutants are sensitive to chlorate in the presence of ammonia or glutamine, whereas the wild type is chlorate resistant under these conditions. a cos ...19882906403
method for identification of intracellular free flavin species in the photosensitive fungus neurospora crassa.establishing the relative intracellular proportions of flavins in neurospora crassa (and in other organisms) in vivo may be hampered by degradation of flavins after homogenization of the cells. the system described here allows separation and identification of intracellular free and bound flavins under conditions restrictive for the fad-degrading enzyme(s). a "protective buffer" containing 0.1 m citrate adjusted to ph 4.0 with k2hpo4, 5 mm atp, and 0.5 mm edta prevents fad from rapid enzymatic cl ...19882973261
growth regulation by gtp. regulation of nucleotide pools in neurospora by nitrogen and sulfur control systems.purine nucleotide pools in the fungus neurospora crassa decline in response to carbon, nitrogen, or sulfur deprivation. there is, in addition, a decline in gtp/atp ratios on nitrogen or sulfur deprivation in wild type. the gtp/atp decline is missing on nitrogen deprivation of the nitrogen control mutant, nit-2, and on sulfur deprivation of the sulfur control mutant, cys-3. the nit-2 mutant also shows elevated utp pools on nitrogen deprivation when compared with similarly treated wild type. six-h ...19882969890
molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase.the metabolic status of a patient previously characterized as deficient in sulfite oxidase was reexamined applying new methodology which has been developed to distinguish between a defect specific to the sulfite oxidase protein and sulfite oxidase deficiency which arises as a result of molybdenum cofactor deficiency. urothione, the metabolic degradation product of the molybdenum cofactor, was undetectable in urine samples from the patient. analysis of molybdenum cofactor levels in fibroblasts by ...19883219233
cytoplasmic leucyl-trna synthetase of neurospora crassa is not specified by the leu-5 locus.we generated a lambda gt11 neurospora crassa cdna library and screened the library for the cytoplasmic leucyl-trna synthetase (cyto leurs) clones using cyto leurs specific antibody. two clones, lambda nclrsc1 and lambda nclrsc2, were obtained which have inserts of approximately 2 kbp and approximately 1.3 kbp, and which overlap by about 0.6 kbp. the following lines of evidence indicate that lambda nclrsc1 and lambda nclrsc2 encode parts of cyto leurs. (1) antibodies affinity purified using eithe ...19882842224
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
a mutant of neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria.the nuclear cyt-2-1 mutant of neurospora crassa is characterized by a gross deficiency of cytochrome c (bertrand, h., and collins, r. a. (1978) mol. gen. genet. 166, 1-13). the mutant produces mrna that can be translated into apocytochrome c in vitro. apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mrna and convert i ...19882454235
factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes.proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive h(+)-atpase in microsomes from maize (zea mays l.) roots washed with 0.25 molar ki decreased as a function of time at 0 to 4 degrees c. the rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. the decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate-sensitive atp hydrolys ...198816666192
blue light induces circadian rhythms in the bd mutant of neurospora: double mutants bd,wc-1 and bd,wc-2 are blind.this paper describes a new blue light effect for neurospora crassa, the photoinduction of circadian rhythms in the bd mutant. the wc-1 and wc-2 genes are necessary for this effect.19882977186
characterization of a mutation that causes overproduction of inositol in neurospora crassa.slow-growing (inl+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of neurospora crassa that produces defective myo-inositol-1-phosphate synthase (mips), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. the defective enzyme has some residual activity. in the inl+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. the mutation (opi1) responsible for the ...19882975749
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