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polyamine regulation of ornithine decarboxylase synthesis in neurospora crassa.ornithine decarboxylase (odc) of the fungus neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. in n. crassa, polyamines repress the synthesis and increase the degradation of odc protein. changes in the rate of odc synthesis correlate with similar changes in the abundance of spe-1 mrna. we identify two sequence elements, one in each of the 5' and 3' regions of the spe-1 gene of n. crassa, requi ...200010733579
characterization of an unusual trna-like sequence found inserted in a neurospora retroplasmid.we characterized an unusual trna-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of neurospora intermedia strain varkud. we previously identified two forms of the trna-like sequence, one of 64 nt (trl-64) and the other of 78 nt (trl-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear trna intron. here, we show that trl-78 is encoded in varkud mitochondrial (mt)dna within a 7 kb sequence that is ...200010710417
ego-1 is related to rna-directed rna polymerase and functions in germ-line development and rna interference in c. elegans.cell-fate determination requires that cells choose between alternative developmental pathways. for example, germ cells in the nematode worm caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. germ-line mitosis depends on a somatic signal that is mediated by a notch-type signaling pathway. the ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for o ...200010704412
nadh dehydrogenase in neurospora crassa contains myristic acid covalently linked to the nd5 subunit peptide.the mitochondrial, proton-pumping nadh:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex i catalyzes the first steps of mitochondrial electron transfer and proton translocation. radiolabel from [(3)h]myristic acid was incorporated by neurospora crassa into the mitochondrial-encoded, approximately 70 kda nd5 subunit of nadh dehydrogenase, as shown by immunoprecipitation. this myristate apparently was linked to ...200010699461
cryo-electron tomography of neurospora mitochondria.cryo-electron tomography was used to study the structural organization of whole frozen-hydrated mitochondria from neurospora crassa. unlike mitochondria from many other species and tissues, in this case the cristae form a three-dimensional network of interconnected lamellae. basically, the three-dimensional structure of ice-embedded mitochondria from this species is consistent with previous descriptions of mitochondria prepared by chemical fixation and resin embedding. nonetheless, ice-embedded ...200010675296
an osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in neurospora crassa.an osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by repeat induced point mutation (rip) from a cross between a wild-type n. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group ii (lgii). the mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. dna transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. a ...200010660063
a conserved mechanism for post-transcriptional gene silencing?proteins with homology to rna-directed rna polymerases function in post-transcriptional gene silencing: in quelling in the fungus neurospora crassa, rnai in the nematode caenorhabditis elegans, and co-suppression in the mustard plant arabidopsis thaliana. these findings are consistent with a conserved mechanism operating in these diverse species.200011178243
repeat-induced point mutation (rip) in crosses with wild-isolated strains of neurospora crassa: evidence for dominant reduction of rip.seventy-one wild-isolated strains of neurospora crassa were examined for their ability to support repeat-induced point mutation (rip) in the erg-3 locus. rip was exceptionally inefficient but detectable in crosses with the strain fgsc 430 from adiopodoume, ivory coast. we could find no consistent differences in ascospore yields when wild isolates identified as "low-rip" or "high-rip" strains were crossed with strains bearing the segmental duplication dp(iiir > [i; ii])ar17. this suggested that r ...200011170738
a neurospora crassa arp1 mutation affecting cytoplasmic dynein and dynactin localization.of the actin-related proteins, arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. the role of arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. usin ...200011129047
ph homeostasis of the circadian sporulation rhythm in clock mutants of neurospora crassa.the influence of environmental (extracellular) ph on the sporulation rhythm in neurospora crassa was investigated for wild-type (frq+) and the mutants chr, frq1, frq7, and frq8. in all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular ph in the range 4-9. on the other hand, for the same ph range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq1. a loss of ph homeostasis of the period, however, was observ ...200011128290
heat shock protein 80 of neurospora crassa: sequence analysis of the gene and expression during the asexual phase.heat shock protein 80 (hsp80) of neurospora crassa, a member of the stress-90 protein family, is a cytosolic molecular chaperone that interacts directly with hsp70 to form a hetero-oligomeric complex. the complete nucleotide sequence of the gene encoding this protein, along with the 5'- and 3'-flanking dna, is reported. the coding sequence is interrupted by two introns, 61 and 30 nucleotides, respectively, in length. the deduced amino acid sequence corresponds to a 695-residue polypeptide with a ...200011109485
in vivo chitin-cadmium complexation in cell wall of neurospora crassa.fungal cell wall, mainly composed of chitin, an n-acetylglucosamine polymer, is known to participate in heavy metal detoxification. in the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of neurospora crassa. based on the results of physical techniques, such as solid-state 13c-nmr, x-ray diffraction, ir and molecular modeling, a structure was proposed for the chitin-cadmium complex. the ring and c-3 hydroxyl oxygens ...200011099854
hex-1, a gene unique to filamentous fungi, encodes the major protein of the woronin body and functions as a plug for septal pores.we have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or woronin bodies, of neurospora crassa. analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. when grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. the hex-1 protein was predicted to be 19,125 da. analysis of the n- ...200011273682
reversion of acridine mustard-induced ad-3 mutants of neurospora crassa. 20005366005
the radiation sensitivity of nuclease mutants in neurospora crassa. 20005366004
genetic studies of urease mutants in neurospora crassa. 20005796944
comparison of the complementation and genetic maps of closely linked nonallelic markers on linkage group i of neurospora crassa. 20005796943
identification of genetic alterations induced by ethyl methanesulfonate in neurospora crassa. 20005714159
relations between complementation patterns and genetic alterations in nitrous acid-induced ad-3b mutants of neurospora crassa. 20006059024
allelic complementation among nitrous acid-induced ad-3b mutants of neurospora crassa. 20006059023
effects of carbon sources on the levels of fructose 1,6-biphosphathase in neurospora crassa. 2000225772
purification and characterization of flavokinase from neurospora crassa.the atp-dependent phosphorylation of riboflavin to fmn by flavokinase is the key step in flavin biosynthesis. flavokinase has been purified from a fungal source for the first time. the enzyme purified from a cell wall lacking mutant of neurospora crassa, slime, is a monomer of m(r) 35.5 kda with maximal activity at alkaline ph and high temperature (55 degrees c). the k(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nm for riboflavin and 210 nm for mgatp ...199910650711
the goodwin oscillator: on the importance of degradation reactions in the circadian clock.this article focuses on the goodwin oscillator and related minimal models, which describe negative feedback schemes that are of relevance for the circadian rhythms in neurospora, drosophila, and probably also in mammals. the temperature behavior of clock mutants in neurospora crassa and drosophila melanogaster are well described by the goodwin model, at least on a semi-quantitative level. a similar semi-quantitative description has been found for neurospora crassa phase response curves with resp ...199910643743
recombinational landscape across a 650-kb contig on the right arm of linkage group v in neurospora crassa.we have established a 650-kb walk spanning the sp to ure-1 interval on neurospora crassa linkage group v. loci covered by the walk are, from proximal to distal, sp; rec-2; ure-2; dna polymerase delta; am; gul-1; ace-5; and ure-1. we have found extensive dna polymorphism in this region and used this to examine the recombinational landscape. crossovers are not evenly distributed over the region covered by the contig.199910591967
isolation and characterization of neurospora crassa nucleoside diphosphate kinase ndk-1.we have previously reported that phosphorylation of a 15-kda protein increased after blue-light irradiation in neurospora crassa. in this study, the 15-kda protein was purified using four columns; deae-cellulose, blue-sepharose, sp-sepharose and mono q. the 15-kda protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. a gene encoding n. crassa nucleoside diphosphate kinase, ndk-1, was is ...199910583364
the tom core complex: the general protein import pore of the outer membrane of mitochondria.translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane tom complex. we have isolated the tom core complex of neurospora crassa by removing the receptors tom70 and tom20 from the isolated tom holo complex by treatment with the detergent dodecyl maltoside. it consists of tom40, tom22, and the small tom components, tom6 and tom7. this core complex was also purified directly from mitochondria after solubilization with do ...199910579717
dna repair and specific-locus mutagenesis in neurospora crassa. 199910556617
involvement of protein kinase c in the response of neurospora crassa to blue light.as a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. we tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. this approach has implicate ...199910517328
microscopic analysis of neurospora ropy mutants defective in nuclear distribution.movement and distribution of nuclei in fungi has been shown to be dependent on microtubules and the microtubule-associated motor cytoplasmic dynein. neurospora crassa mutants known as ropy are defective in nuclear distribution. we have shown that three of the ro genes, ro-1, ro-3, and ro-4, encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. three other ro genes, ro-7, ro-10, and ro-11, are required for the integrity or localization of cytoplasmic dynein or dy ...199910512672
characterization of a neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.we produced a photolyase-deficient mutant by repeat induced point mutation using the neurospora crassa photolyase gene cloned previously. this mutation identified a new gene, phr, which was mapped on the right arm of linkage group i by both rflp mapping and conventional mapping. to investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and ...199910512668
action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in neurospora.in neurospora crassa, dna sequence duplications are detected and altered efficiently during the sexual cycle by a process known as rip (repeat-induced point mutation). affected sequences are subjected to multiple gc-to-at mutations. to explore the pattern in which base changes are laid down by rip we examined two sets of strains. first, we examined the products of a presumptive spontaneous rip event at the mtr locus. results of sequencing suggested that a single rip event produces two distinct p ...199910511550
characterization of neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase.neurospora crassa cel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding beta-fatty acid synthase. these mutants are phenotypically less leaky than cel-1, which has a defective alpha-fatty acid synthase. the cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and synthesized less fatty acid de novo than cel-1. unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for f ...199910501937
inactivation of the neurospora crassa mitochondrial outer membrane protein tom70 by repeat-induced point mutation (rip) causes defects in mitochondrial protein import and morphology.mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the tom complex (translocase of the outer mitochondrial membrane). the largest component of this complex is tom70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. to investigat ...199910501936
a reductase/isomerase subunit of mitochondrial nadh:ubiquinone oxidoreductase (complex i) carries an nadph and is involved in the biogenesis of the complex.respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping nadh:ubiquinone oxidoreductase, or complex i. the bacterial complex i consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. one of these extra subunits with a molecular mass of 40 kda belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. we deleted this subun ...199910497022
effects of disrupting the 21 kda subunit of complex i from neurospora crassa.we have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kda subunit of complex i from neurospora crassa. mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex i in approximately normal amounts. in addition, this mutant is able to assemble an almost intact enzyme. the electron transfer activities from nadh to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type s ...199910477266
induced revertants of biochemical mutants of neurospora crassa: personal recollections. 199910610431
learning to "think like a geneticist" with the de serres ad-3 forward-mutation test in neurospora crassa. 199910610427
the antibiotic viomycin as a model peptide for the origin of the co-evolution of rna and proteins.viomycin is an rna-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group i intron self-splicing. this antibiotic enhances the activity of the ribozyme derived from the neurospora crassa vs rna, and at sub-inhibitory concentrations it induces the formation of group i intron oligomers. here, we address the question whether viomycin exerts specificity in the promotion of rna-rna interactions. in an in vitro selection experiment we tested the ability of viomycin to specif ...199910472628
gene silencing: rna makes rna makes no protein.a mutation that disrupts post-transcriptional gene silencing in neurospora crassa has been found to affect the homologue of a plant-encoded rna-dependent rna polymerase. this enzyme may produce a specificity determinant of gene silencing and mediate an epigenetic conversion at the rna level.199910469584
nucleotide binding and hydrolysis properties of neurospora crassa cytosolic molecular chaperones, hsp70 and hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.formation of a hetero-oligomeric complex between hsp70 and hsp80 of neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (elisa). the present study documents the effect of nucleotides on the subunit structure of hsp70 and hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (dma), and dimethyl suberimidate (dms). the inter-protomer crosslinking of hsp80 with dma and dms was suppressed by atp and to a ...199910438143
the drosophila melanogaster gene for the nadh:ubiquinone oxidoreductase acyl carrier protein: developmental expression analysis and evidence for alternatively spliced forms.we have isolated the drosophila melanogaster gene encoding the mitochondrial acyl carrier protein (mtacp), a subunit of nadh:ubiquinone oxidoreductase involved in de novo fatty acid synthesis in the mitochondrion. this gene expresses two distinct mature transcripts by alternative splicing, which encode mature polypeptides of 86 (mtacp1a) and 88 (mtacp1b) amino acids, respectively. drosophila mtacp1 is 72% identical to mammalian mtacp, 47% identical to arabidopsis thaliana mtacp, and 46% identica ...199910394906
the nop-1 gene of neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. previously, genes encoding opsins had been identified in animals and the archaea but not in fungi or other eukaryotic microorganisms. here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus neurospora crassa. the nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid ...199910393943
evidence for negative interference: clustering of crossovers close to the am locus in neurospora crassa among am recombinants.in response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, fincham proposed that recombination events at the neurospora am locus rarely have an associated crossover. fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am i ...199910388816
assignment of circadian function for the neurospora clock gene frequency.circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. it is difficult to assign molecular clock components to any one of these elements. experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator. mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the p ...199910376598
neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution.movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. we have isolated hundreds of neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. in this report, we describe the isolation and initial characterization o ...199910361308
the structure of the vacuolar atpase in neurospora crassa.the filamentous fungus neurospora crassa contains many small vacuoles. these organelles contain high concentrations of polyphosphates and basic amino acids, such as arginine and ornithine. because of their size and density, the vacuoles can be separated from other organelles in the cell. the atp-driven proton pump in the vacuolar membrane is a typical v-type atpase. we examined the size and structure of this enzyme using radiation inactivation and electron microscopy. the vacuolar atpase is a la ...199910340846
gene silencing in neurospora crassa requires a protein homologous to rna-dependent rna polymerase.in plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. this phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. transgene-induced gene silencing has been termed quelling in neurospora crassa and co-suppression in plants. quelling-defective (qde) mutants of n. crassa, in which transgene-induced gene silencing is ...199910335848
functional analysis of the two zinc fingers of sre, a gata-type factor that negatively regulates siderophore synthesis in neurospora crassa.multiple gata factors, zinc finger dna binding proteins that recognize consensus gata elements, exist in neurospora crassa. one of them, sre, is involved in controlling the iron metabolic pathway of n. crassa. in n. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. the siderophore synthesis pathway is negatively regulated by sre; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. in the research presented here, ...199910194352
glucose metabolism in neurospora is altered by heat shock and by disruption of hsp30.we compared the metabolism of [1-13c]glucose by wild type cells of neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. high temperature led to increased incorporation of 13c into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees c). heat shock strongly reduced formation of tricarboxylic a ...199910076052
microconidia of neurospora crassa.neurospora crassa produces two types of vegetative spores-relatively small numbers of uninucleate microconidia and very large numbers of multinucleate macroconidia (blastoconidia and arthroconidia). the microconidia can function either as spermatia (male gametes) or as asexual reproductive structures or both. in nature they probably function exclusively in fertilization of protoperithecia. the environmental conditions favoring their formation and the pattern of their development are quite distin ...199910072316
the goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of neurospora crassa.the goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of neurospora and drosophila. an essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. when protein synthesis in n. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (chx) the period of the circadian sporulation rhythmicity ...199910036201
the b regulatory subunit of protein phosphatase 2a is required for completion of macroconidiation and other developmental processes in neurospora crassa.rgb-1, encoding the tentative b regulatory subunit of the type 2a ser/thr phosphatase in neurospora crassa, was isolated from cdna and genomic libraries. based on analysis of cdna and genomic clones, rgb-1 is 3387 nucleotides in length, contains seven putative introns and encodes a 461-amino-acid polypeptide. intron i, which is 5' to the presumed translation initiation codon, contains a uorf encoding 34 amino acids. intron vi undergoes alternative splicing. inactivation of rgb-1 by the repeat-in ...19999987122
molecular characterization of tol, a mediator of mating-type-associated vegetative incompatibility in neurospora crassa.the mating-type locus in the haploid filamentous fungus, neurospora crassa, controls mating and sexual development. the fusion of reproductive structures of opposite mating type, a and a, is required to initiate sexual reproduction. however, the fusion of hyphae of opposite mating type during vegetative growth results in growth inhibition and cell death, a process that is mediated by the tol locus. mutations in tol are recessive and suppress mating-type-associated heterokaryon incompatibility. i ...19999927450
functional anatomy of the kinesin molecule in vivo.we have developed an assay that allows the functional efficiency of mutant kinesins to be probed in vivo. we show here that the growth rate of the filamentous fungus neurospora crassa can be used as a sensitive reporter for the ability of mutant kinesins to suppress the phenotype of the kinesin null mutant of neurospora. truncation mutants, internal deletion mutants and chimeras, in which homologous domains were exchanged between different fungal kinesins, were generated and transformed into the ...199910449406
radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.a few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. the enzyme isoorotate decarboxylase (idcase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. we describe in this communication two assays for idcase and the ...19999887216
mutational activation of a galphai causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in neurospora crassa.heterotrimeric g proteins, consisting of alpha, beta, and gamma subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. we previously reported that the filamentous fungus neurospora crassa possesses a galpha protein, gna-1, that is a member of the galphai superfamily. deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. in addition, deltagna-1 strains have low ...19999872952
cloning and sequencing of the chromosomal dna and cdna encoding the mitochondrial citrate synthase of aspergillus niger wu-2223l.the complementary dna (cdna) and chromosomal dna encoding the citrate synthase (ec 4.1.3.7) gene (cit1) of aspergillus niger wu-2223l, a citric acid-producing strain, were cloned. synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of a. niger genes. the 920-bp dna fragment was amplified by polymerase chain reaction with these primers using chromosomal dna of wu-2223l as a template, and was employe ...199916232605
neurospora crassa glutamine synthetase. role of enzyme synthesis and degradation on the regulation of enzyme concentration during exponential growth.the specific activity of neurospora crassa glutamine synthetase varies according to the nitrogen source in which the organism is grown. in a poor nitrogen source such as glutamate, the specific activity of the enzyme is higher than that found in good nitrogen sources such as ammonium or glutamine. these differences in specific enzyme activity correspond to differences in enzyme concentration. the relative rates of glutamine synthetase synthesis and degradation were measured in exponential cultur ...199921883
affinity chromatography of the neurospora nadp-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.the synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-sepharose 4b, is described. the assembly of the 2'-amp ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the sepharose matrix. this adsorbent retarded the hexameric nadp-specific glutamate dehydrogenase of neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. the enzyme was eluted e ...199922328
the mycoparasite ampelomyces quisqualis expresses exga encoding an exo-beta-1,3-glucanase in culture and during mycoparasitism.abstract ampelomyces quisqualis, a mycoparasite of fungi causing powdery mildews, exhibited high levels of extracellular exo-beta-1,3-glucanase activity in culture compared with neurospora crassa and gliocladium roseum. a. quisqualis culture filtrates affected powdery mildew caused by sphaerotheca fusca in a manner indicative of cell wall degradation, as determined by microscopic examination. a gene encoding an exo-beta-1,3-glucanase in a. quisqualis, designated exga, was isolated and sequenced. ...199918944674
comparative analysis of ca2+ and h+ flux magnitude and location along growing hyphae of saprolegnia ferax and neurospora crassa.calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete saprolegnia ferax and the ascomycete neurospora crassa and pseudohyphal saccharomyces cerevisiae using self-referencing ion-selective probes. s. ferax exhibited well-defined transport zones absent in n. crassa. ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net ca2+ flux was either inward (75% of all experiments) or outward. the inward component of the net flux was inhib ...199910669108
the frost gene of neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis.the neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding ca(2+), suggesting that characterization of this gene might clarify the mechanism of ca(2+)-dependent tip growth. the wild-type allele was cloned by sib selection using protoplasts from arthroconidia. rflp analysis revealed that the cloned dna fragment mapped to the fr locus. the nucleotide sequence of genomic and cdna was determined. the deduced amino acid sequence showed homology to the saccharomy ...199910669587
use of atp6 in fungal phylogenetics: an example from the boletales.complete nucleotide sequences have been determined for atp6 from suillus luteus and cox3 from suillus sinuspaulianus (boletales, hymenomycetes, basidiomycota), which code for atpase subunit 6 and cytochrome oxidase subunit 3, respectively. these sequences were used to design pcr primers for the amplification of partial atp6 and cox3 sequences from other members of the boletales and outgroup taxa. in atp6 and cox3 from russula rosacea, one of the outgroup taxa, we observed a number of in-frame tg ...199910620406
cloning and characterization of a gene (arpa) from aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores.we have isolated the arpa gene from aspergillus oryzae as a homologue of the neurospora crassa ro-4 gene. in n. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex arp1, causes curling of hyphae and abnormalities in nuclear distribution. the arpa gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the n. crassa arp1. overexpression of the arpa gene causes a defect in nuclear migration into elongating hyph ...199910628858
in vitro antifungal evaluation and studies on the mode of action of xanthoxyline derivatives.this study describes the fungistatic effect of xanthoxyline (cas 90-24-4) and its derivatives against a panel of yeasts, filamentous fungi and dermatophytes, by using the agar dilution method. results indicated that simple structural modifications led to more potent derivatives, especially in relation with dermatophytes. the most active compound tested (10), which is a benzenesulphonyl derivative, was 12-fold more potent than xanthoxyline itself against trichophyton rubrum. the evaluation of the ...199910635452
posttranscriptional gene silencing in neurospora by a recq dna helicase.the phenomenon of posttranscriptional gene silencing (ptgs), which occurs when a transgene is introduced into a cell, is poorly understood. here, the qde-3 gene, which is required for the activation and maintenance of gene silencing in the fungus neurospora crassa, was isolated. sequence analysis revealed that the qde-3 gene belongs to the recq dna helicase family. the qde3 protein may function in the dna-dna interaction between introduced transgenes or with an endogenous gene required for gene- ...199910600745
a highly conserved mechanism of regulated ribosome stalling mediated by fungal arginine attenuator peptides that appears independent of the charging status of arginyl-trnas.the arg attenuator peptide (aap) is an evolutionarily conserved peptide involved in arg-specific negative translational control. it is encoded as an upstream open reading frame (uorf) in fungal mrnas specifying the small subunit of arg-specific carbamoyl phosphate synthetase. we examined the functions of the saccharomyces cerevisiae cpa1 and neurospora crassa arg-2 aaps using translation extracts from s. cerevisiae, n. crassa, and wheat germ. synthetic rna containing aap and firefly luciferase ( ...199910608810
similarity pattern analysis in mutational distributions.the validity and applicability of the statistical procedure - similarity pattern analysis (span) - to the study of mutational distributions (mds) was demonstrated with two sets of data. the first was mutational spectra (ms) for 697 gc to at transitions produced with eight alkylating agents (aas) in the laci gene of escherichia coli. the second was a recently summarized data on the distributions of 11562 spontaneous, radiation- and chemical-induced forward mutations in the ad-3 region of heteroka ...199910592318
disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of neurospora crassa, causes defects in import of proteins into mitochondria.the gene for hsp30, the only known alpha-crystallin-related heat shock protein of neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. hsp30, which is not synthesized at 30 degrees c, associates reversibly with the mitochondria at high temperature (45 degrees c). in this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired ...199910595587
chitin synthase genes in the arbuscular mycorrhizal fungus glomus versiforme: full sequence of a gene encoding a class iv chitin synthase.chitin synthase genes of the arbuscular mycorrhizal fungus glomus versiforme were sought in an investigation of the molecular basis of fungal growth. three dna fragments (gvchs1, gvchs2 and gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (pcr) with two sets of degenerate primers. gvchs1 and gvchs2 encode two class i chitin synthases, whereas gvchs3 encodes a class iv chitin synthase. a genomic library ...19999919652
cloning and expression of the cdna encoding an alternative oxidase gene from aspergillus niger wu-2223l.a cdna fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (sham)-sensitive respiration, from the citric acid-producing fungus aspergillus niger wu-2223l was cloned and expressed in escherichia coli as a host strain. synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. the 210-bp dna fragment was amplified by pcr with these primers usi ...19999933359
the aspergillus nidulans gata factor srea is involved in regulation of siderophore biosynthesis and control of iron uptake.a gene encoding a new gata factor from aspergillus nidulans, srea, was isolated and characterized. srea displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to sre from neurospora crassa and about 50% identity to urbs1 from ustilago maydis over a stretch of 200 amino acid residues containing two gata-type zinc finger motifs and a cysteine-rich region. this putative dna binding domain, expressed as a fusion protein in escherichia coli, specifically bi ...19999988696
cloning of two genes encoding potassium transporters in neurospora crassa and expression of the corresponding cdnas in saccharomyces cerevisiae.two neurospora crassa genes, trk-1 and hak-1, encode k+ transporters that show sequence similarities to the trk transporters described in saccharomyces cerevisiae and schizosaccharomyces pombe, and to the hak transporters described in schwanniomyces occidentalis and barley. the n. crassa trk1 and hak1 transporters expressed by the corresponding cdnas in a trk1 delta trk2 delta mutant of s. cerevisiae exhibited a high affinity for rb+ and k+. northern blot analysis and comparison of the kinetic c ...199910027968
the wd protein cpc2p is required for repression of gcn4 protein activity in yeast in the absence of amino-acid starvation.the cpc2 gene of the budding yeast saccharomyces cerevisiae encodes a g beta-like wd protein which is involved in regulating the activity of the general control activator gcn4p. the cpc2 gene encodes a premrna which is spliced and constitutively expressed in the presence or absence of amino acids. loss of cpc2 gene function suppresses a deletion of the gcn2 gene encoding the general control sensor kinase, but not a deletion in the gcn4 gene. the resulting phenotype has resistance against amino-a ...199910048025
isolation, characterization and disruption of the area nitrogen regulatory gene of gibberella fujikuroi.the gene area-gf, a homologue of the major nitrogen regulatory genes nit-2, area, nre and nut1 of neurospora crassa, aspergillus nidulans, penicillium chrysogenum and magnaporthe grisea, respectively, was cloned from the gibberellin (ga)-producing rice pathogen gibberella fujikuroi. area-gf encodes a protein of 972 amino acid residues which contains a single putative zinc finger dna-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. the ar ...199910071216
positive regulation of adenylyl cyclase activity by a galphai homolog in neurospora crassa.gna-1 and gna-2 are two g protein alpha subunits from the filamentous fungus neurospora crassa. loss of gna-1 leads to multiple phenotypes, while deltagna-2 strains do not exhibit visible defects. however, deltagna-1deltagna-2 mutants are more affected in deltagna-1 phenotypes. here we report a biochemical investigation of the roles of gna-1 and gna-2 in camp metabolism. assays of mg2+ atp-dependent adenylyl cyclase activity (+/-gppnhp) in extracts from submerged cultures indicated that deltagna ...199910072319
pyruvate decarboxylase filaments are associated with the cortical cytoskeleton of asci and spores over the sexual cycle of filamentous ascomycetes.we show that pyruvate decarboxylase (pdc) 8- to 10-nm-diameter filaments, first described in vegetative cells of neurospora crassa, are ubiquitously present in filamentous fungi. the cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes n. crassa, n. tetraesperma, podospora anserina, and sordaria macrospora. pdc-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and sp ...199910072321
amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and 4.pit2 is the human receptor for amphotropic murine leukemia virus (a-mulv); the related human protein pit1 does not support a-mulv entry. interestingly, chimeric proteins in which either the n-terminal or the c-terminal part of pit2 was replaced by the pit1 sequence all retained a-mulv receptor function. a possible interpretation of these observations is that pit1 harbors sequences which can specify a-mulv receptor function when presented in a protein context other than pit1, e.g., in pit1-pit2 h ...199910074169
physiological consequence of disruption of the vma1 gene in the riboflavin overproducer ashbya gossypii.the vacuolar atpase subunit a structural gene vma1 of the biotechnologically important riboflavin overproducer ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the vma1 proteins from yeast and filamentous fungi. the deduced polypeptide comprises 617 amino acids with a calculated ...199910092625
the ukc1 gene encodes a protein kinase involved in morphogenesis, pathogenicity and pigment formation in ustilago maydis.the fungal phytopathogen ustilago maydis alternates between budding and filamentous growth during its life cycle. this dimorphic transition, which is influenced by environmental factors and mating, is regulated in part by camp-dependent protein kinase (pka). we have recently identified a related protein kinase, encoded by the ukc1 gene, that also plays a role in determining cell shape. the ukc1 gene is homologous to several other protein kinase-encoding genes including the cot-i gene of neurospo ...199910102363
characterisation of the last fe-s cluster-binding subunit of neurospora crassa complex i.we have cloned cdnas encoding the last iron-sulphur protein of complex i from neurospora crassa. the cdna sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 da. our results indicate that the mature protein belongs probably to the peripheral arm of complex i and is rather unstable when not assembled into the enzyme. the protein is highly homologous to the psst subunit of bovine complex i, the most likely candida ...199910216160
copper- and zinc-containing superoxide dismutase and its gene from candida albicans.cytosolic copper- and zinc-containing superoxide dismutase was purified 136-fold with an overall yield of 2.5% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, candida albicans. the molecular mass of the native enzyme was 39.4 kda and the enzyme was composed of two identical subunits with a molecular mass of 19.6 kda. the enzyme was stable in the range of ph 4.0-9.0 and up to 55 degrees c. the ultraviolet-visible absorption spectrum of the enzyme showed the absorptio ...199910216241
a common ancestor for a subunit in the mitochondrial proton-translocating nadh:ubiquinone oxidoreductase (complex i) and short-chain dehydrogenases/reductases.the proton-translocating nadh:ubiquinone oxidoreductase or complex i is located in the inner membranes of mitochondria, where it catalyzes the transfer of electrons from nadh to ubiquinone. here we report that one of the subunits in complex i is homologous to short-chain dehydrogenases and reductases, a family of enzymes with diverse activities that include metabolizing steroids, prostaglandins and nucleotide sugars. we discovered that a subunit of complex i in human, cow, neurospora crassa and ...199910228558
neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi aspergillus nidulans and neurospora crassa.a camp-activatable ca2+-dependent neutral trehalase was identified in germinating conidia of aspergillus nidulans and neurospora crassa. using a pcr approach, a. nidulans and n. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. disruption of the antreb gene encoding a. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is part ...199910320571
nad+-dependent glutamate dehydrogenase of the edible mushroom agaricus bisporus: biochemical and molecular characterization.the nad+-dependent glutamate dehydrogenase (nad-gdh) of agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity. the apparent molecular mass of the native enzyme is 474 kda comprising four subunits of 116 kda. the isoelectric point of the enzyme is about 7.0. km values for ammonium, 2-oxoglutarate, nadh, glutamate and nad+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mm, respectively. the enzyme is specific for nad(h). the gene encoding this enzyme (gdhb) was isolated from an ...199910323225
interaction of mitochondrial presequences with dnak and mitochondrial hsp70.mitochondrial heat shock protein 70 (mt-hsp70) functions as a molecular chaperone in mitochondrial biogenesis. the chaperone in co-operation with its co-proteins acts as a translocation motor pulling the mitochondrial precursor into the matrix. mt-hsp70s are highly conserved when compared to the bacterial hsp70 homologue, dnak. here we have used dnak as a model to study the interaction of mitochondrial presequences with mt-hsp70 applying a dnak-binding algorithm, computer modeling and biochemica ...199910329135
signal presequences increase mitochondrial permeability and open the multiple conductance channel.we have reported that the signal presequence of cytochrome oxidase subunit iv from neurospora crassa increases the permeability of isolated rat liver mitochondria [p. m. sokolove and k. w. kinnally (1996) arch. biochem. biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (mcc) of yeast inner mitochondrial membrane [t. a. lohret and k. w. kinnally (1995) j. biol. chem. 270, 15950]. here we examine in greater detail the action of a number of mitochondrial presequences f ...199910334870
molecular cloning and characterization of ct-pkar, a gene encoding the regulatory subunit of camp-dependent protein kinase in colletotrichum trifolii.colletotrichum trifolii is a plant pathogenic fungus causing alfalfa anthracnose. prepenetration development, including conidial germination and appressorial formation, are requisite for successful infection. pharmacological data from our laboratory indicated a role for a camp-dependent protein kinase (pka) pathway during these early morphogenic transitions. thus, the cloning and characterization of the genes for pka catalytic and regulatory subunits were undertaken to more precisely determine t ...199910339807
molecular mechanisms of chromosomal rearrangement in fungi.both sexual and asexual fungi undergo chromosomal rearrangements, which are the main cause of karyotype variability among the populations. different recombination processes can produce chromosomal reorganizations, both during mitosis and meiosis, but other mechanisms operate to limit the extent of the rearrangements; some of these mechanisms, such as the rip (repeat-induced point mutations) of neurospora crassa, have been well established for sexual fungi. in laboratory strains, treatments such ...199910342097
carbon regulation of ribosomal genes in neurospora crassa occurs by a mechanism which does not require cre-1, the homologue of the aspergillus carbon catabolite repressor, crea.transcription of the ribosomal protein and 40s rrna genes is coordinately regulated during steady state growth and carbon shifts in neurospora crassa. recognition sequences for the aspergillus nidulans carbon catabolite repressor, crea, overlap transcriptional elements of a 40s rrna gene and the crp-2 ribosomal protein gene. they also occur in similar locations in the promoters of several other ribosomal protein genes. substitutions encompassing the -74 and -167 crea consensus sequences in the c ...199910361038
identification of a 24 kda intrinsic membrane protein from mammalian peroxisomes.a 24 kda protein from rat liver peroxisomal membrane was isolated and subjected to edman degradation. using the n-terminal sequence of this polypeptide we have identified several rat and human expressed sequence tags in the genbank database. the complete sequence of a human cdna clone was determined. the open reading frame encodes an extremely basic protein 212 amino acid residues long. a high similarity between this mammalian protein and hypothetical proteins from caenorhabditis elegans and neu ...199910366717
hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.neurospora crassa arg13 and saccharomyces cerevisiae arg11 encode mitochondrial carrier family (mcf) proteins that transport ornithine across the mitochondrial inner membrane. we used their sequences to identify est candidates that partially encode orthologous mammalian transporters. we thereby identified such a gene (ornt1) that maps to 13q14 and whose expression, similar to that of other urea cycle (uc) components, was high in liver and varied with changes in dietary protein. ornt1 expression ...199910369256
pagrg1, a glucose-repressible gene of podospora anserina that is differentially expressed during lifespan.a loss-of-function mutation in grisea, a gene of podospora anserina that was previously shown to code for a copper-modulated transcription factor, leads to a significant increase in lifespan. in an attempt to identify and to isolate potential target genes controlled by grisea, a rt-differential-display screen was performed. this approach resulted in the identification of a gene, pagrg1, that is differentially expressed in the wild-type and in the long-lived grisea mutant. in the mutant, transcri ...199910369964
a single external enzyme confers alternative nadh:ubiquinone oxidoreductase activity in yarrowia lipolytica.nadh:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial nadh oxidation. in addition to the energy-conserving form commonly called complex i, fungi and plants contain much simpler alternative nadh:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. little is known about the distribution and function of these enzymes. we have identified ylndh2 as the only gene encoding an al ...199910381390
assaying rna chaperone activity in vivo using a novel rna folding trap.in the absence of proteins, rnas often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. we identify an in vivo folding trap in the t4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. during translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. the introduction of a stop ...199910393192
transcription of the avirulence gene avr9 of the fungal tomato pathogen cladosporium fulvum is regulated by a gata-type transcription factor in aspergillus nidulans.the avirulence gene avr9 of the fungal tomato pathogen cladosporium fulvum is highly induced during infection of tomato plants. expression of the avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. the avr9 promoter contains six copies of the sequence tagata and six additional copies of the core sequence gata within 0.4 kb upstream of the translation start site. in the filamentous fungi aspergillus nidulans and neurospora crass ...199910394902
comparison of h+-atpase and ca2+-atpase suggests that a large conformational change initiates p-type ion pump reaction cycles.structures have recently been solved at 8 a resolution for both ca2+-atpase from rabbit sarcoplasmic reticulum and h+-atpase from neurospora crassa. these cation pumps are two distantly related members of the family of p-type atpases, which are thought to use similar mechanisms to generate atp-dependent ion gradients across a variety of cellular membranes. we have undertaken a detailed comparison of the two structures in order to describe their similarities and differences as they bear on their ...199910395538
singlet oxygen is part of a hyperoxidant state generated during spore germination.we show that singlet oxygen is generated in asexual spores (conidia) from neurospora crassa at the onset of germination. oxidation of n. crassa catalase-1 (cat-1) was previously shown to be caused by singlet oxygen (lledías et al. j. biol. chem. 273, 1998). in germinating conidia, increased protein oxidation, decrease of total protein, cat-1 oxidation and accumulation of cat-1 mrna was detected. these changes were modulated in vivo by light intensity, an external clean source of singlet oxygen, ...199910401603
flocculation of hyphae is associated with a deletion in the putative cahk1 two-component histidine kinase gene from candida albicans.in candida albicans, three putative histidine kinase genes have been described thus far, including casln1, canik1/cos1 and cahk1. the encoded proteins for c. albicans, casln1p and canik1p, which are similar to sln1p from saccharomyces cerevisiae and nik-1 from neurospora crassa, seem to function in osmoregulation and morphogenesis, respectively. recently, the isolation of cahk1, a putative histidine kinase gene from c. albicans has been reported. in addition to the histidine and aspartyl domains ...199910411270
the erg-3 (sterol delta14,15-reductase) gene of neurospora crassa: generation of null mutants by repeat-induced point mutation and complementation by proteins chimeric for human lamin b receptor sequences.null mutations were generated in the erg-3 gene of neurospora crassa by repeat-induced point mutation (rip). the mutants were viable, lacked ergosterol, were resistant to the steroidal glycoside alpha-tomatine and were sensitive to the phytoalexins pisatin and biochanin a. rip was frequently associated with silencing of the hph gene located adjacent to the duplicated erg-3 sequence. the silencing of hph was reversible in the two cases examined and appeared to be due to the spread of cytosine met ...199910411271
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