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cytosine methylation associated with repeat-induced point mutation causes epigenetic gene silencing in neurospora crassa.repeated dna sequences are frequently mutated during the sexual cycle in neurospora crassa by a process named repeat-induced point mutation (rip). rip is often associated with methylation of cytosine residues in and around the mutated sequences. here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. a large proportion of strains that had undergone rip of a linked duplication flanking a single-copy transgene, hph (hygromycin b phosphotransferase), showed ...19979178002
rco-3, a gene involved in glucose transport and conidiation in neurospora crassa.macroconidiation in neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. conidia formation is normally observed when the fungus is exposed to air. however, carbon limitation can induce conidiation in mycclia submerged in an aerated liquid medium. a mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been clon ...19979178001
novel pattern of dna methylation in neurospora crassa transgenic for the foreign gene hph.it has previously been reported that multiple copies of the hph gene integrated into the genome of neurospora crassa are methylated at hpa ii sites (ccgg) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. furthermore, methylation is correlated with silencing of the gene. we report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy str ...19979171093
expression of herpes virus thymidine kinase in neurospora crassa.the expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of dna metabolism and provide useful drug-sensitive phenotypes. the herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in neurospora crassa. tk was expressed as a fusion to n.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and sv40 sequences. only strains containing tk showed thymidi ...19979171090
cloning and characterization of a cdna encoding a bacteriophage-type rna polymerase from the higher plant chenopodium album.we have cloned a full-length cdna from the higher plant chenopodium album coding for a single subunit bacteriophage-type rna polymerase. the cdna isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on northern blots. the open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kda. a comparison of the protein sequence with those of the two known fungal mitochondrial rna polymerases, from saccharomyces cerevisiae and neurospora crass ...19979171081
melanogenesis, tyrosinase expression, and reproductive differentiation in black and white truffles (ascomycotina).white and black truffles of the genus tuber are ascomycotina as well as neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. we present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. l-tyrosine 3-monooxygenase and l-dopa oxidase activities colocalize as histochemically detected and are simi ...20169170162
mutations of pma-1, the gene encoding the plasma membrane h+-atpase of neurospora crassa, suppress inhibition of growth by concanamycin a, a specific inhibitor of vacuolar atpases.concanamycin a (cca), a specific inhibitor of vacuolar atpases, inhibited growth of neurospora crassa in medium adjusted to ph 7 or above. mutant strains were selected for growth on medium containing 1.0 microm cca. sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane h+-atpase. analysis of v-atpase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to cca. in contrast, plasma membrane h+-atp ...19979169444
a putative role for calmodulin in the activation of neurospora crassa chitin synthase.the possible role of calmodulin in cell wall formation and chitin synthesis was studied in neurospora crassa by examining the effects of anti-calmodulin agents on protoplast regeneration and possible associations between chitin synthase and calmodulin related proteins in microsomal isolates. protoplast regeneration was inhibited by trifluoperazine (> 20 microm), an anticalmodulin agent. chitin synthase activity in microsomes was associated with that of calmodulin-dependent protein kinase and inh ...19979163912
molecular cloning and expression studies of two divergent alpha-tubulin genes in neurospora crassa.three alpha-tubulin isoforms were previously detected in neurospora crassa. we have cloned and analysed two alpha-tubulin cdnas, tub alpha a and tub alpha b that encode polypeptides of 453 and 451 amino acids, respectively. the encoded amino acids exhibit an unusual divergence of 35%. this is the highest divergence ever observed between alpha-tubulins from the same species. the expression of the two genes is developmentally regulated. we did not detect any transcription of the tub alpha a gene i ...19979163903
cloning of an e. coli reca and yeast rad51 homolog, rada, an allele of the uvsc in aspergillus nidulans and its mutator effects.an e. coli reca and yeast rad51 homolog from aspergillus nidulans, rada, has been cloned by screening genomic and cdna libraries with a pcr-amplified probe. this probe was generated using primers carrying the conserved sequences of eukaryotic reca homologs. the deduced amino acid sequence revealed two conserved walker-a and -b type nucleotide-binding domains and exhibited 88%, 60%, and 53% identity with mei-3 of neurospora crassa, rhp51+ of schizosaccharomyces pombe, and rad51 of saccharomyces c ...19979163746
purification and characterization of an antifungal pr-5 protein from pumpkin leaves.a 28-kda antifungal pr-5 protein (pltp) was purified from pumpkin leaves to homogeneity by using ammonium sulfate fractionation, a regenerated chitin column, and reversed-phase column chromatographies on butyl-toyopearl and hplc c18 columns. analysis of 14 n-terminal amino acid sequences of pltp shows 100% sequence identity to those of two pr-5 proteins, np24 from tomatoes and ap24 from tobacco. the identical sequence also exhibited high amino acid sequence homology to that of an osmotin-like pr ...19979163735
identification of putative phosphoinositide-specific phospholipase c genes in filamentous fungi.five putative phosphoinositide-specific phospholipases c (plc) genes were identified in three species of filamentous fungi. using polymerase chain reaction with degenerate oligonucleotide primers, gene fragments encoding amino acid sequences homologous to plcs of mammals and other organisms were amplified: one sequence from botryotinia fuckeliana, one from aspergillus nidulans, and three from neurospora crassa. the molecular cloning and sequencing of a putative plc gene (bcplc1) from b. fuckelia ...19979163731
a gene required for nuclear migration in neurospora crassa codes for a protein with cysteine-rich, lim/ring-like domains.the ro mutants of neurospora crassa are defective in nuclear migration and hyphal morphogenesis. several of the ro loci have recently been shown to encode components of the dynein/dynactin motor complex. here we report on the cloning and characterization of the ro-2 gene which codes for a novel 80 kda protein that has two cys-rich motifs which resemble zinc-binding lim or ring domains thought to mediate protein-protein interactions. ro2 also contains several potential binding sites for src homol ...19979159520
evidence for and characterization of cytochrome p-450 in neurospora crassa.cytochrome p-450 was detected in microsomes and presumably in cytosol of neurospora crassa, and was found to be inducible by progesterone. in the microsomal fraction cytochrome b5 and nadph-cytochrome c reductase activities were measurable, too. cytochrome p-450 of neurospora crassa is inhibited by skf-525 a and by inhibitors of ergosterol biosynthesis. after induction of cytochrome p-450 with progesterone 11 alpha-hydroxyprogesterone as one metabolite of progesterone was detected in the culture ...19979151426
[energy and metabolic intercellular interaction in the growth region of neurospora crassa]. 19979147633
the osmotic-1 locus of neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast.osmotically sensitive mutants of neurospora crassa are unable to grow on medium supplemented with 4% nacl, have altered morphologies and cell-wall compositions, and are resistant to dicarboximide fungicides. osmotic-1 (os-1) mutants have a unique characteristic of forming protoplasts that grow and divide in specialized liquid medium, suggesting that the os-1+ gene product is important for cell-wall assembly. a cosmid containing the os-1+ locus of n. crassa, isolated from a genomic cosmid library ...19979142740
cloning of cutinase transcription factor 1, a transactivating protein containing cys6zn2 binuclear cluster dna-binding motif.hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at -159 base pairs of the cutinase gene in fusarium solani f. sp. pisi (nectria hematococca mating type vi). of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids. screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinc ...19979139694
mutation of the cys-9 gene, which encodes thioredoxin reductase, affects the circadian conidiation rhythm in neurospora crassa.ten cysteine auxotrophs of neurospora crassa were examined with regard to the period lengths of their circadian conidiation rhythms. one of the these cysteine auxotrophs, cys-9, showed dramatic changes in the circadian conidiation rhythm. at 10 microm methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. at this concentration of methionine, the period length was unstable after the fo ...19979136004
quantitative and qualitative comparison of spontaneous and chemical-induced specific-locus mutation in the ad-3 region of heterokaryon 12 of neurospora crassa.the data from forward-mutation experiments to obtain specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (h-12) of neurospora crassa have been tabulated to determine the relative frequencies and mutational spectra of ad-3 mutants occurring spontaneously and those induced by 22 different chemical treatments. previous studies have demonstrated that specific-locus mutations at these two loci result from 5 major genotypic classes, namely two classes ...19979129679
quantitative and qualitative comparisons of spontaneous and radiation-induced specific-locus mutation in the ad-3 region of heterokaryon 12 of neurospora crassa.the data from forward-mutation experiments to obtain specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (h-12) of neurospora crassa have been used to determine the relative frequencies and mutational spectra of ad-3 mutants occurring spontaneously and those induced by 7 different radiation treatments. previous studies have demonstrated that specific-locus mutants at these two loci result from 5 major genotypic classes, namely two classes of gene ...19979129678
efficient cloning of ascomycete mating type genes by pcr amplification of the conserved mat hmg box.cloning of mating type (mat) genes from ascomycetes has been hampered by low conservation among them. one of the pair of mat genes, represented by mat-2 of cochliobolus heterostrophus (a loculoascomycete) and mt a of neurospora crassa (a pyrenomycete), encodes a protein with a conserved dna binding motif called the high mobility group (hmg) box. pcr with primer pairs corresponding to the borders of the c. heterostrophus and the n. crassa hmg boxes generated an approximately 0.3-kb product from g ...19979126621
developmental and photoregulation of three neurospora crassa carotenogenic genes during conidiation induced by desiccation.neurospora crassa asexual sporulation (conidiation) is induced by different cues including desiccating aerial environments. three of the genes that are expressed during this developmental pathway are the albino (al) genes, which encode carotenoid biosynthetic enzymes. if conidiation is induced by nutrient deprivation in liquid culture, two overlapping al-3 mrnas, al-3 (m) and al-3 (c), are expressed (arpaia et al., 1995, dev. biol. 170, 620-635). here we quantitate accumulation of each of the fo ...19979126619
cross-pathway and pathway-specific control of amino acid biosynthesis in magnaporthe grisea.the gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, arg2, of magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen. the transcript of the arg2 gene contains an upstream open reading frame (uorf) that is similar to uorfs found in the homologous genes of neurospora crassa (arg-2) and saccharomyces cerevisiae (cpa1), suggesting that the m. grisea gene is translationally regulated by a mechanism that ...19979126616
purification of a 47-kda calmodulin-binding polypeptide as an actin-binding protein from neurospora crassa.we have enriched a 47-kda polypeptide (p47) from neurospora crassa on the basis of its affinity to calmodulin. the p47 was purified to homogeneity by chromatography on a mono s cation exchange column and evidence is presented that the polypeptide co-sediments specifically with f-actin. the intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. our results suggest that by altering the conformatio ...19979119196
mutants of neurospora crassa defective in regulation of blue light perception.a new selection system was used to isolate mutants deficient in regulation of blue light perception in neurospora crassa. this selection system has two possible applications and was used for the isolation of either blind or constitutive mutants. we isolated 17 uv-induced mutants that showed the pleiotropic white collar (wc) phenotype and were completely blocked in transduction of the light signal. from the segregation pattern in sexual crosses, we tentatively assigned the 17 mutants to either th ...19979108272
identification of genes induced in alkaloid-producing cultures of claviceps sp.in order to identify genes which are expressed during alkaloid synthesis in an axenic culture of claviceps sp. (strain atcc 26245), a cdna library from a producing culture was differentially screened with cdna from producing (cdna+) and non-producing (cdna-) cultures, respectively. altogether, ten cdna clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by northern analyses. evaluation of their nucleotide and derived amino-acid sequences identified one gene ...19979108144
phospholipid composition of highly purified mitochondrial outer membranes of rat liver and neurospora crassa. is cardiolipin present in the mitochondrial outer membrane?isolated mitochondrial outer membrane vesicles (omv) are a suitable system for studying various functions of the mitochondrial outer membrane. for studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. recently, a mild subfractionation procedure was described for the isolation of highly purified omv from mitochondria of neurospora crassa (may ...19979106488
genetic regulation of nitrogen metabolism in the fungi.in the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the gata family of proteins. however, certain gata family members, such as the yeast dal80 fact ...19979106362
mutagenic activity and specificity of hydrogen peroxide in the ad-3 forward-mutation test in two-component heterokaryons of neurospora crassa.in the ad-3 forward-mutation test, hydrogen peroxide was at best a weak mutagen in nongrowing conidia from a dna repair-proficient heterokaryon (h-12, uvs-2+/uvs-2+) but was a moderate mutagen in nongrowing conidia from a dna-repair-deficient heterokaryon (h-59, uvs-2/uvs-2) over a narrow range of high concentrations. h-59 also was more sensitive than h-12 to the killing activity of hydrogen peroxide at high concentrations. thus, a dna-repair pathway, of which the gene product of the uvs-2+ alle ...19979100841
inhibition of neurospora crassa growth by a glucan synthase-1 antisense construct.we have used the filamentous fungus, neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-glucan synthase [e.c. 2.4.1.34; udpglucose: 1,3-beta-d-glucan 3-beta-d-glucosyltransferase], leads to a corresponding decrease in growth of the organism. previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucan synthase activity. wild-type cells were transformed with dna vectors encodin ...19979099631
effect of nonylphenol on growth of neurospora crassa and candida albicans.the effects of the nonionic surfactant nonylphenol on the growth and morphologies of the filamentous fungus neurospora crassa and the diploid yeast candida albicans have been examined. nonylphenol inhibited respiration and growth of n. crassa, effecting a 10-fold decrease in organism yield at 25 microm. severe morphological defects were also induced: cell shape was abnormal and apical dominance was lost. nonylphenol monoethoxylate (the parent compound of nonylphenol) was a less potent growth inh ...19979097428
transport of small lons and molecules through the plasma membrane of filamentous fungi.less than 1% of the estimated number of fungal species have been investigated concerning the transport of low-molecular-weight nutrients and metabolites through the plasma membrane. this is surprising if one considers the importance of the processes at the plasma membrane for the cell: this membrane mediates between the cell and its environment. concentrating on filamentous fungi, in this review emphasis is placed on relating results from biophysical chemistry, membrane transport, fungal physiol ...19979097013
pathways for fatty acid elongation and desaturation in neurospora crassa.neurospora crassa incorporated exogenous deuterated palmitate (16:0) and 14c-labeled oleate (18:1 delta 9) into cell lipids. of the exogenous 18:1 delta 9 incorporated, 59% was desaturated to 18:2 delta 9,12 and 18:3 delta 9,12,15. of the exogenous 16:0 incorporated, 20% was elongated to 18:0, while 37% was elongated and desaturated into 18:1 delta 9, 18:2 delta 9,12, and 18:3 delta 9,12,15. the mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass ...19979075186
efficient cloning of ascomycete mating type genes by pcr amplification of the conserved mat hmg boxcloning of mating type (mat) genes from ascomycetes has been hampered by low conservation among them. one of the pair of mat genes, represented by mat-2 of cochliobolus heterostrophus (a loculoascomycete) and mt a of neurospora crassa (a pyrenomycete), encodes a protein with a conserved dna binding motif called the high mobility group (hmg) box. pcr with primer pairs corresponding to the borders of the c. heterostrophus and the n. crassa hmg boxes generated an approximately 0.3-kb product from g ...19979073486
developmental and photoregulation of three neurospora crassa carotenogenic genes during conidiation induced by desiccationneurospora crassa asexual sporulation (conidiation) is induced by different cues including desiccating aerial environments. three of the genes that are expressed during this developmental pathway are the albino (al) genes, which encode carotenoid biosynthetic enzymes. if conidiation is induced by nutrient deprivation in liquid culture, two overlapping al-3 mrnas, al-3 (m) and al-3 (c), are expressed (arpaia et al., 1995, dev. biol. 170, 620-635). here we quantitate accumulation of each of the fo ...19979073484
cross-pathway and pathway-specific control of amino acid biosynthesis in magnaporthe griseathe gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, arg2, of magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen. the transcript of the arg2 gene contains an upstream open reading frame (uorf) that is similar to uorfs found in the homologous genes of neurospora crassa (arg-2) and saccharomyces cerevisiae (cpa1), suggesting that the m. grisea gene is translationally regulated by a mechanism that ...19979073479
characterization of neurospora mitochondrial group i introns reveals different cyt-18 dependent and independent splicing strategies and an alternative 3' splice site for an intron orf.the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) functions in splicing the n. crassa mitochondrial large rrna intron by stabilizing the catalytically active structure of the intron core. here, a comprehensive study of n. crassa mtdna group i introns identified two additional introns, cob-i2 and the nd1 intron, that are dependent on cyt-18 for splicing in vitro and in vivo. the other seven n. crassa mtdna group i introns are not cyt-18-dependent and include five that s ...19979042940
analysis of frequency (frq) clock gene homologs: evidence for a helix-turn-helix transcription factor.the frq gene plays a key role in the organization of the neurospora crassa circadian clock. our previous analysis of a c-terminal fragment of the putative frq protein suggested it is a nuclear transcription factor but did not identify a known dna-binding domain. if the hypothesis is correct that frq is a transcription factor, sequences consistent with this function should be conserved in distantly related species. to investigate, we have cloned frq homologs from other filamentous fungi including ...19979037100
repeat-induced point mutations of hsp80 gene of neurospora crassa: methylation of duplicated dna sequences in the vegetative state.the process of repeat-induced point mutations (rip) was used to disrupt the gene encoding the 80-kda heat-inducible protein of neurospora crassa. germinated conidia of the wild-type recipient strain were electrotransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. some of the transformants with a duplication of hsp80 gene sequence showed extensive methylation of these sequences even in vegetatively growing cells. the presence of an extra gene copy in ...19969035689
disruption of the nad(+)-specific glutamate dehydrogenase gene of neurospora crassa by means of the rip (repeat-induced point mutations) process.the structural gene for the catabolite-repressed, substrate-induced nad(+)-specific glutamate dehydrogenase (gdh-1) of neurospora crassa was disrupted using the process of repeat-induced point mutation (rip). plasmids containing incomplete copies of the gene, along with selectable markers, were introduced into germinated conidia by electroporation. the sexual progeny of a transformant containing an ectopically integrated copy of a plasmid, harbouring the 5' flanking region and a part of the codi ...19969035688
three-dimensional structure of nadh-dehydrogenase from neurospora crassa by electron microscopy and conical tilt reconstruction.nadh-dehydrogenase (complex i) is the first complex of the mitochondrial respiratory chain. it is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear dna. the whole complex was isolated in detergent from the fungus neurospora crassa. it is very stable in its isolated form and was analysed as such by electron microscopy. its mass, determined by dark-field scanning electron microscopy was estima ...19979034360
serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from neurospora crassa.the major spontaneously active serine/threonine (ser/thr) protein phosphatase activities in n. crassa wild type (fgsc 424) were type-1 (pp1), type-2a (pp2a) and type-2c (pp2c). pp1 and pp2c predominantly dephosphorylated phosphorylase a and casein, respectively. pp2a acted on both substrates, but was two-fold more active against casein. pp1 activity was inhibited by protamine, heparin, okadaic acid (ic50 50 nm) and mammalian inhibitor-1 (ic50 2 nm). on the other hand. pp2a activity was inhibited ...19969033809
cloning and characterization of a neurospora crassa ribosomal protein gene, crps-7.a neurospora crassa ribosomal protein gene, crps-7, was isolated from a genomic dna library closely linked to the morphological gene ro-2. sequence analysis and a computerized database search revealed a high degree of homology to the xenopus laevis rps8 and rat rps7 gene, as well as to uncharacterized orfs from two yeast species. comparison with a nearly full-length cdna clone revealed two introns, one of which is in a conserved position shared with the xenopus gene. although a number of sequenc ...19979021131
white collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in neurospora crassa.a saturating genetic dissection of 'blind' mutants in neurospora crassa has identified a total of two non-redundant loci (wc-1 and wc-2) each of which is required for blue-light perception/signal transduction. previously, we demonstrated that wc1 is a putative zinc finger transcription factor able to bind specifically to a light-regulated promoter. here, we present the cloning and characterization of the wc-2 gene. we demonstrate using mutation analysis and in vitro dna-binding assays that wc2, ...19979009271
the aspergillus niger gcn4 homologue, cpca, is transcriptionally regulated and encodes an unusual leucine zipper.the general control transcriptional regulator gene cpca of aspergillus niger was cloned by complementation of a saccharomyces cerevisiae delta gcn4 mutant strain. the encoded protein conferred resistance to amino acid analogues when expressed in yeast. disruption of cpca in a. niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation, cpca encodes a transcript of approximately 2400 nucleotides in length that includes a 5' leader region of ...19979004217
subunit vii of ubiquinol:cytochrome-c oxidoreductase from neurospora crassa is functional in yeast and has an n-terminal extension that is not essential for mitochondrial targeting.cdna clones encoding subunit vii of the neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit viii of the complex from yeast (encoded by qcr8), were identified on the basis of functional complementation of a yeast qcr8 deletion strain. the clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kda. the n-terminal eight residues of the amino acid sequence deduced from the cdna clones are absent from the ma ...19969003361
functional promoter elements common to ribosomal protein and ribosomal rna genes in neurospora crassa.the promoter sequences of a cytoplasmic ribosomal protein gene (crp-2) of neurospora crassa were identified using promoter deletion and substitution mutants. a gene-targeting strategy was used to assay the mutants in vivo. the promoter architecture of crp-2 is complex and is more similar to that of ribosomal protein genes in mouse than in saccharomyces cerevisiae. six regions were identified as important for transcription. these included two elements, a cg repeat and a dde box, that are conserve ...19969003305
arginine-specific regulation mediated by the neurospora crassa arg-2 upstream open reading frame in a homologous, cell-free in vitro translation system.translational control mediated by an upstream open reading frame (uorf) in the 5'-leader of the neurospora crassa arg-2 mrna was reconstituted in a homologous, cell-free in vitro translation system. a cell-free n. crassa system was developed that required the presence of cap and poly(a) on rna for maximal translation and that was amino acid-dependent. the 24-codon arg-2 uorf, when placed in the 5'-leader region of capped and adenylated synthetic luciferase rnas, conferred arg-specific negative r ...19978995256
metabolism of ricinoleate by neurospora crassa.neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. one such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. to develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). we therefore examined effects of ricinoleate on lipid biosynthesis and growth of n. crassa. ricinoleate ...19968987727
cloning and functional analysis of a beta-tubulin gene from a benomyl resistant mutant of aspergillus parasiticus.a genomic dna library prepared from a benomyl resistant strain of aspergillus parasiticus was screened with a neurospora crassa beta-tubulin gene probe. a unique a. parasiticus genomic dna fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. two plasmids, pyt1 and pytpyrg, carrying the putative benr gene or benr plus a second selectable marker (pyrg), respectively, were used to transform a benomyl sensitive strain of a. parasiticus (cs10) to determine if benr conferred ben ...19968982061
isolation and characterization of a copper containing protein from blue cell walls of neurospora crassa.culturing neurospora crassa in presence of toxic amounts of copper (0.63 mm) resulted in blue coloured mycelia and cell walls. significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. while decuprification of the blue cell walls with 8-hydroxy quinoline (8 hq) did not alter their copper binding abilities, similar t ...19968979504
a tyrosyl-trna synthetase recognizes a conserved trna-like structural motif in the group i intron catalytic core.the neurospora crassa mitochondrial (mt) tyrosyl-trna synthetase (cyt-18 protein) functions in splicing group i introns, in addition to aminoacylating trna(tyr). here, we compared the cyt-18 binding sites in the n. crassa mt lsu and nd1 introns with that in n. crassa mt trna(tyr) by constructing three-dimensional models based on chemical modification and rna footprinting data. remarkably, superimposition of the cyt-18 binding sites in the model structures revealed an extended three-dimensional o ...19968978617
a dominant negative effect of eth-1r, a mutant allele of the neurospora crassa s-adenosylmethionine synthetase-encoding gene conferring resistance to the methionine toxic analogue ethionine.eth-1r, a thermosensitive allele of the neurospora crassa s-adenosylmethionine (adomet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. replacement of an aspartic amino acid residue (d48-->n48), perfectly conserved in prokaryotic, fungal and higher eukaryotic adomet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells r ...19968978034
saccharomyces cerevisiae chorismate synthase has a flavin reductase activity.chorismate synthase (cs) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (epsp) to form chorismate, which is the last common intermediate in the synthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. despite the overall redox-neutral reaction, catalysis has an absolute requirement for reduced flavin. in the fungus neurospora crassa, a flavin reductase (fr) activity able to generate reduced flavin mononucleotide in the presence of nadph is an intrinsic ...19968971708
the chsb gene of aspergillus nidulans is necessary for normal hyphal growth and development.the chsb gene from aspergillus nidulans encodes a class iii chitin synthase, an enzyme class found in filamentous fungi but not in yeast-like organisms. using a novel method, we isolated haploid segregants carrying a disrupted chsb allele from heterozygous diploid disruptants. the haploid disruptants grow as minute colonies that do not conidiate. hyphae from the disruptants have enlarged tips, a high degree of branching, and disorganized lateral walls. the mycelium is not deficient in chitin con ...19968953267
translocation of neurospora crassa transcription factor nuc-1 into the nucleus is induced by phosphorus limitation.nuc-1, a basic helix-loop-helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in neurospora crassa. in the present study we investigated whether posttranscriptional mechanisms control the activity of nuc-1. the nuc-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosph ...19968953266
a mitochondrial signal peptide from neurospora crassa increases the permeability of isolated rat liver mitochondria.mitochondria that contain ca2+ can be induced by a variety of triggering agents and conditions to undergo a permeability transition (pt); the inner membrane becomes nonselectively permeable to small solutes. mastoparan, an amphipathic peptide from wasp venom, has recently been reported to induce this transition (pfeiffer et al., 1995, j. biol. chem. 270,4923). we have examined the effect on the permeability of isolated rat liver mitochondria of a second amphipathic peptide, the signal sequence o ...19968951036
cloning and sequencing of the lys1 gene encoding homocitrate synthase in the yeast yarrowia lipolytica.the alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. the first step in the pathway is the condensation of acetyl-coa and alpha-ketoglutarate into homocitrate, and this step is carried out by the enzyme homocitrate synthase (ec 4.1.3.21). in spite of extensive genetic analysis, no mutation affecting this step has been isolated until now in model organisms such as saccharomyces cerevisiae or neurospora crassa, although identification of mutations affe ...19968948100
acetylglutamate synthase from neurospora crassa: structure and regulation of expression.a dna clone which complemented an arg-14 mutation of neurospora crassa was isolated by sib selection from a cosmid library (pmocosx). southern and restriction-fragment-length polymorphism (rflp) analysis confirmed that the cloned dna contained the arg-14 gene. the arg-14 gene was identified as the structural gene for acetylglutamate synthase by immunodepletion of enzyme activity with antibodies prepared against an arg-14 fusion protein and by the thermal instability of acetylglutamate synthase i ...19968939437
blue light induced adp ribosylation of 38 and 56 kda proteins in the soluble fraction of mycelia of neurospora crassa.soluble fractions prepared from the mycelia of wild type (74-or23-1a) and band (bd) exhibited an increase in the rate of the adp ribosylation of a 38 kda protein from nicotinamide adenine [32p]dinucleotide ([32p]nad) in the presence of 10(-7) m riboflavin caused by blue light irradiation in vitro. the soluble fraction was mixed with a reaction mixture containing 5 microci [32p]nad at 0 degree c for 20 s and then it was irradiated with blue light (420 nm, 42 mumol m-2 s-1) for 12.5, 25, 50, 100, ...19968933725
phosphate transporters from the higher plant arabidopsis thaliana.two cdnas (atpt1 and atpt2) encoding plant phosphate transporters have been isolated from a library prepared with mrna extracted from phosphate-starved arabidopsis thaliana roots, the encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of saccharomyces cerevisiae, neurospora crassa, and the mycorrhizal fungus glomus versiforme. the atpt1 and atpt2 polypeptides are integral membrane proteins predicte ...19968927627
intracellular phosphate--water oxygen exchange measured by mass spectrometry.to study, in vivo, potential p(i)-water oxygen exchange catalyzed by each of two high-affinity p(i) symporters of neurospora crassa, we have developed methods for the purification of p(i) from whole-cell extracts and the subsequent derivatization of p(i) for analysis by gc-ms. we have also modified a published procedure for the preparation of 18o-p(i). however, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of 18o-p(i) in cells incubate ...19968921158
loss of growth polarity and mislocalization of septa in a neurospora mutant altered in the regulatory subunit of camp-dependent protein kinase.in filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. to explore this process, we analyzed a conditional neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. cloning and dna sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of camp-dependent protein kinase (pka). unexpectedly, the mcb mutant still formed sept ...19968918454
circadian clock-controlled genes isolated from neurospora crassa are late night- to early morning-specific.an endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. in the fungus neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. to understand more fully the processes that are regulated by the n. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. time-of-day-specific cdn ...19968917550
nuc-2, a component of the phosphate-regulated signal transduction pathway in neurospora crassa, is an ankyrin repeat protein.in response to phosphorus limitation, the fungus neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. the nuc-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. the nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high ph and low phosphate concentration. we mapped the cloned gene to the right arm of linkage group ii, ...19968917314
role of cytosolic serine hydroxymethyltransferase in one-carbon metabolism in neurospora crassa.conidiospores of wild type and two mutant strains of neurospora crassa were grown on [3-13c]serine, [2-13c]glycine, or [13c]formate. acid extracts of the mycelia were analyzed by 13c nmr for incorporation of the 13c label into choline, serine, and adenine. the goal was to elucidate the function of cytosolic serine hydroxymethyltransferase by comparison of a mutant strain lacking this activity and requiring formate for optimal growth (for mutant strain) to the wild-type strain and the ser3 strain ...19968914930
isolation and characterization of a laccase gene from podospora anserina.the genome of the filamentous ascomycete podospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene of neurospora crassa. one of these regions contains a gene (lac2) encoding a protein that displays 62% identity with the n. crassa laccase. in shaken cultures, lac2 mrna is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. addition of aromatic xen ...19968914515
asm-1+, a neurospora crassa gene related to transcriptional regulators of fungal development.this report describes the identification, cloning, and molecular analysis of asm-1+ (ascospore maturation 1), the neurospora crassa homologue of the aspergillus nidulans stua (stunted a) gene. the asm-1+ gene is constitutively transcribed and encodes an abundant, nucleus-localized 68.5-kd protein. the protein product of asm-1+ (asm-1), contains a potential dna-binding motif present in related proteins from a. nidulans (stua), candida albicans (efgtf-1), and saccharomyces cerevisiae (phd1 and sok ...19968913744
structural investigation of a cell-wall galactomannan from neurospora crassa and n. sitophila. 19968901273
identification of the native nit2 major nitrogen regulatory protein in nuclear extracts of neurospora crassa.the nit-2 gene of neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. an e. coli-expressed nit2/beta-gal fusion protein binds specifically to dna in vitro by recognizing gata core elements. nuclear extracts prepared from a wild-type n. crassa strain contain a protein factor which displays all of the properties expected for the native nit2 protein. t ...19968901135
functional in vivo studies of the neurospora crassa cys-14 gene upstream region: importance of cys3-binding sites for regulated expression.sulphate transport in neurospora crassa is achieved by two distinct sulphate permeases, i and ii, encoded by the cys-13 and cys-14 genes, respectively. the synthesis of both sulphate permeases is subject to sulphur repression and requires the global positive-acting regulatory protein cys3, cys3, a bzip dna binding protein, regulates cys-14 expression at the transcriptional level and binds in vitro specifically to three dna-recognition sites, a, b, and c, in the cys-14 upstream region. in vivo fu ...19968899713
evolution of the frequency (frq) clock locus in ascomycete fungi.the frequency (frq) locus of neurospora crassa plays a key role in the organization of circadian rhythms. similar timing systems have been found in nearly all eukaryotes as well as some prokaryotes; thus, frq may be an excellent gene with which to conduct evolutionary studies. to investigate, we used the cloned frq locus from ascomycete fungi representing two classical taxonomic classes and three orders to examine two open questions in ascomycete evolution. class pyrenomycetidae is represented b ...19968896376
escape from het-6 incompatibility in neurospora crassa partial diploids involves preferential deletion within the ectopic segment.self-incompatible het-6or/het-6pa partial diploids of neurospora crassa were selected from a cross involving the translocation strain, t(iil-->iiir)ar18, and a normal sequence strain. about 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that "escape" from het-6 incompatibility had occurred. near isogenic tester strains with different alleles (het-6or and het-6pa) were constructed and used to determine that 80 of 96 escape strains tested were het- ...19968889517
characterization of rco-1 of neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of tup1 of saccharomyces cerevisiae.the filamentous fungus neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. selection was based on expression of an integrated dna fragment containing the con-10 promoter-regulatory reg ...19968887652
m-glycogenin, the protein moiety of neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme.neurospora crassa proteoglycogen was purified and its protein moiety, m-glycogenin, was released by amylolytic treatment. the released protein was capable of autoglucosylation from udp-glucose forming glucosyl-alpha 1,4-glucosyl linkage. the kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. m-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. both auto- and transglucosylation reactions were dependen ...19968886029
mild rip-an alternative method for in vivo mutagenesis of the albino-3 gene in neurospora crassa.we have used a biological phenomenon that occurs in neurospora crassa, termed repeat-induced point mutation (rip), to create partially functional mutant alleles of the albino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. a total of 70 rip-induced al-3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid bio-synthesis. nucleotide sequence analysis of ...19968879235
light-induced phase shifting of the circadian conidiation rhythm is inhibited by calmodulin antagonists in neurospora crassa.the effects of calmodulin antagonists and inhibitors of protein kinases and phosphatases on light-induced phase shifting were investigated in neurospora crassa. calmodulin antagonists, namely, trifluoperazine, chlorpromazine, and w-7, almost completely inhibited the light-induced phase shifting without having any effect on the circadian clock itself. chlorpromazine was less effective in inhibiting the light-induced phase shifting than trifluoperazine. w-5, a dechlorinated analogue of w-7, failed ...19968872595
nuva, an aspergillus nidulans gene involved in dna repair and recombination, is a homologue of saccharomyces cerevisiae rad18 and neurospora crassa uvs-2.a 40 kb genomic clone and 2.3 kb ecori subclone that rescued the dna repair and recombination defects of the aspergillus nidulans nuva11 mutant were isolated and the subclone sequenced. the subclone hybridized to a cosmid in a chromosome-specific library confirming the assignment of nuva to linkage group iv and indicating its closeness to bimd. amplification by pcr clarified the relative positions of nuva and bimd. a region identified within the subclone, encoding a c3hc4 zinc finger motif, was ...19968868425
repeat-induced gene silencing: common mechanisms in plants and fungi.one of the most surprising observations made in plant science in recent years is the inactivation of transgenes triggered by interactions between dna repeats. in plants, we can differentiate between transcriptional silencing, most likely reflecting a regulation at the dna level, and post-transcriptional silencing that affects steady state rna levels. in the filamentous fungi ascobolus immersus and neurospora crassa, we find two premeiotic silencing processes that are also based on the interactio ...19968868065
in vivo calcineurin crystals formed using the baculovirus expression system.calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated t cells. coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. the crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclo ...19968859891
the g alpha i homologue gna-1 controls multiple differentiation pathways in neurospora crassa.heterotrimeric g proteins are components of principal signaling pathways in eukaryotes. in higher organisms, alpha subunits of g proteins have been divided into four families, gi, gs, gq, and g12. we previously identified a g alpha i homologue gna-1 in the filamentous fungus neurospora crassa. now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in n. crassa. on solid medium, delta gna-1 strains have a slower rate of hyphal apical extension th ...19968856670
activator-independent gene expression in neurospora crassa.a transgenic position effect that causes activator-independent gene expression has been described previously for three neurospora crassa phosphate-repressible genes. we report analogous findings for two additional positively regulated genes, qa-2+ and ars-1+, indicating that such position effects are not limited to genes involved in phosphorus metabolism. in addition, we have characterized a number of mutants that display activator-independent gene expression. each of these mutants contains a ch ...19968852841
eth-1, the neurospora crassa locus encoding s-adenosylmethionine synthetase: molecular cloning, sequence analysis and in vivo overexpression.intense biochemical and genetic research on the eth-1r mutant of neurospora crassa suggested that this locus might encode s-adenosylmethionine synthetase (s-adomet synthetase). we have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1r mutation to clone the locus. nucleotide sequence analysis demonstrated that it encodes s-adomet synthetase. homology analyses of prokaryotic, fungal and higher eukaryotic s-adomet synthetase polypeptide s ...19968849888
purification and characterization of cytochrome c oxidase from the insect trypanosomatid crithidia fasciculata.cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. the purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of cua in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but ...19968844671
the product of the het-c heterokaryon incompatibility gene of neurospora crassa has characteristics of a glycine-rich cell wall protein.filamentous fungi are capable of hyphal fusion, but heterokaryon formation between different isolates is controlled by specific loci termed het loci. heterokaryotic cells formed between strains of different het genotype are rapidly destroyed or strongly inhibited in their growth. in neurospora crassa, at least 11 loci, including the mating type locus, affect the capacity to form a heterokaryon between different isolates. in this report, we describe the molecular characterization of the vegetativ ...19968844148
development of a metallothionein based heavy metal biosorbent.the potential utility of a recombinant e. coli expressing the neurospora crassa metallothionein gene (ncp) as a heavy metal biosorbent was investigated. it was shown that the ncp was capable of efficiently removing low levels of several metals (including cadmium, lead, and mercury) from solutions. the reusability of the ncp was demonstrated through 5 cycles of metal binding, stripping with dilute acid, and regeneration of the binding sites with out any adverse effect on the metal binding activit ...19968843348
circular dichroism studies of the mitochondrial channel, vdac, from neurospora crassa.the protein that forms the voltage-gated channel vdac (or mitochondrial porin) has been purified from neurospora crassa. at room temperature and ph 7, the circular dichoism (cd) spectrum of vdac suspended in octyl beta-glucoside is similar to those of bacterial porins, consistent with a high beta-sheet content. when vdac is reconstituted into phospholipid liposomes at ph 7, a similar cd spectrum is obtained and the liposomes are rendered permeable to sucrose. heating vdac in octyl beta-glucoside ...19968842216
bli-4, a gene that is rapidly induced by blue light, encodes a novel mitochondrial, short-chain alcohol dehydrogenase-like protein in neurospora crassa.blue light plays an important role in developmental control throughout nature. the bli-4 gene of neurospora crassa, together with bli-3, al-1 and al-2, is rapidly inducible by blue light. induction leads to a ninety-fold increase in transcription rate over the dark control level, and the gene therefore appears to be of prime importance in the blue-light induction pathway of n. crassa. we describe the sequencing and analysis of bli-4 and the 38 kda protein it encodes. we show that the protein is ...19968842141
isolation of laccase gene-specific sequences from white rot and brown rot fungi by pcr.degenerate primers corresponding to the consensus sequences of the copper-binding regions in the n-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. all except three gave the expected pcr product of about 200 bp. computer searches of the databases identified the sequence of each of the pcr products analyzed as a laccase gene sequence, suggesting the specificity of the primers. pcr product ...19968837429
a tyrosyl-trna synthetase suppresses structural defects in the two major helical domains of the group i intron catalytic core.the neurospora crassa mitochondrial tyrosyl-trna synthetase, the cyt-18 protein, functions in splicing group i introns by promoting the formation of the catalytically active structure of the intron rna. the group i intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of p5, p4, p6, and p6a (p4-p6 domain) and the other consisting of p8, p3, p7, and p9 (p3-p9 domain). to investigate how cyt-18 stabilizes the active rna structure, we used an es ...19968831782
genetic variability among isolates and sexual offspring of the plant pathogenic fungus calonectria morganii on the basis of random amplification of polymorphic dna (rapd) and restriction fragment length polymorphism (rflp)thirty-two strains of the phytopathogenic mold cylindrocladium scoparium (perfect state calonectria morganii) isolated from ericaceous hosts and two specimens from the atcc were examined by random amplification of polymorphic dna (rapd) and restriction fragment length polymorphism (rflp). five oligonucleotides were chosen as primers for differentiation of the isolates. rapd patterns of the atcc strains differed significantly from those of the field isolates. diversity among field isolates was lo ...19968824171
properties of a polynucleotide synthesized by strain 74a of neurospora crassa.a polynucleotide (or a fragment of rna) was purified to apparent homogeneity by hplc from mycelium of the wild strain 74a of the mould neurospora crassa, after growth on sucrose and in the presence of saturating amounts of inorganic phosphate (pi) for 72 hr at 30 degrees. the m(r) was ca 20,000 as determined by hplc at ph 6.8. polynucleotide synthesis ranged from 4.0 to 6.5 micrograms polynucleotide per mg dry mycelium in mycelium of the wild strain 74a and the various phosphorus regulatory and ...19968821430
the isolation of a dol-p-man synthase from ustilago maydis that functions in saccharomyces cerevisiae.genomic dnas from several fungi were screened for a homologous sequence to saccharomyces cerevisiae dpm1, an essential gene which encodes dolichyl phosphoryl mannose synthase. the fungi examined included aspergillus nidulans, neurospora crassa, schizophyllum commune and ustilago maydis. only u. maydis gave a significant signal after southern hybridization using dpm1 as a probe. the ustilago homolog was subsequently cloned and sequenced. the predicted protein of 294 amino acids has 60% identity t ...19968813763
a transposon insertion in the arabidopsis ssr16 gene causes an embryo-defective lethal mutation.the ssr16 gene of arabidopsis has been identified as a gene encoding a ribosomal protein s16 homolog through analysis of a transposon insertion mutation. the insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that ...19968811862
covalent attachment of fad derivatives to a fusion protein consisting of 6-hydroxy-d-nicotine oxidase and a mitochondrial presequence. folding, enzyme activity, and import of the modified protein into yeast mitochondria.autoflavinylation of 6-hydroxy-d-nicotine oxidase (6-hdno) was successfully employed to modify the protein covalently with fad derivatives. the model compounds n6-(2-aminoethyl)-fad and n6-(6-carboxyhexyl)-fad were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of neurospora crassa f0-atpase subunit 9 (su9) attached to 6-hdno. when translated in the rabbit reticulocyte lysate, su9-6-hdno was in the trypsin-sensitive apoenzyme form; when translated in t ...19968810280
mutants with altered sensitivity to a calmodulin antagonist affect the circadian clock in neurospora crassa.two newly isolated mutant strains of neurospora crassa, cpz-1 and cpz-2, were hypersensitive to chlorpromazine with respect to mycelial growth but responded differently to the drug with respect to the circadian conidiation rhythm. in the wild type, chlorpromazine caused shortening of the period length of the conidiation rhythm. pulse treatment with the drug shifted the phase and inhibited light-induced phase shifting in neurospora. by contrast to the wild type, the cpz-2 strain was resistant to ...19968807291
isolation and analysis of the arg-13 gene of neurospora crassa.mutations in arg-13 result in slow growth in minimal medium and can suppress mutations in carbamyl phosphate synthase-aspartate carbamyl transferase within the pyrimidine pathway; the exact biochemical function of the gene product is unknown. to understand the role of arg-13 in arginine metabolism, cosmids rescuing growth in arg-13 mutants were cloned and mapped to the position of arg-13 on lg ir. northern analysis showed the arg-13 message to contain approximately 2100 nt, although a 1.4-kb gen ...19968807290
evidences for adenine nucleotide binding in the subunits of neurospora mitochondrial processing peptidase.the mitochondrial processing peptidase (mpp) of neurospora crassa consists of two subunits termed alpha-and beta-mpp. here we present spectroscopic and chromatographic studies indicative of adenine nucleotide binding in the two mpp subunits. adp was identified as the cofactor of alpha-mpp and atp as the cofactor of beta-mpp. the nucleotides are not covalently bound and exert strong control on the conformational and functional properties of the subunits. the adp cofactor of alpha-mpp seems to be ...19968806742
catalytic mechanism of mitochondrial processing peptidase: fluorescence studies.processing of nuclear-encoded precursor proteins by mitochondrial processing peptidase (mpp) is an essential step for their sorting and function in mitochondria. we report spectroscopic studies on the catalytic mechanism of neurospora crassa mpp. it is a complex enzyme consisting of two different subunits termed alpha-and beta-mpp. following changes in the protein intrinsic fluorescence we register and characterize a complex formation between (i) the alpha- and the beta-subunit of mpp, (ii) the ...19968806741
disruption of the nuclear gene encoding the 20.8-kda subunit of nadh: ubiquinone reductase of neurospora mitochondria.the nuclear gene coding for the 20.8-kda subunit of the membrane arm of respiratory chain nadh: ubiquinone reductase (complex i) from neurospora crassa, nuo-20.8, was localized on linkage group i of the fungal genome. a genomic dna fragment containing this gene was cloned and a duplication was created in a strain of n. crassa by transformation. to generate rip (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic mar ...19968804391
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