deoxyribonucleoside triphosphate pools in neurospora crassa: effects of histidine and effective hplc method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus neurospora crassa has been developed. in rapidly growing cells the nucleotide levels vary from 11.8 pmoles/micrograms dna for dgtp to 24.2 pmoles/micrograms dna for dttp. these levels fall by approximately one half in stationary-phase cultures but the ratio of each pool to dgtp remains the same. the dntp pools in conidia are at least 5-fold lower than in rapidly growing cells. the pool sizes are th ...20132969078
mitochondrial protein import: identification of processing peptidase and of pep, a processing enhancing protein.transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. we have isolated the processing activity from neurospora crassa. the final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (mpp, 57 kd) and a processing enhancing protein (pep, 52 kd). the two components were isolated as monomers. pep is about 15-fold more abundant in ...19882967109
molecular analysis of a neurospora crassa gene expressed during conidiation.the asexual developmental pathway in the life cycle of the filamentous fungus neurospora crassa culminates in the formation of spores called conidia. several clones of genomic neurospora dna have been isolated that correspond to mrna species expressed during conidiation and not during mycelial growth (v. berlin and c. yanofsky, mol. cell. biol. 5:849-855, 1985). in this paper we describe the characterization of one of these clones, named pcon-10a. this clone contains two genes, con-10 and con-13 ...19882970007
relationship of histidine sensitivity to dna damage and stress induced responses in mutagen sensitive mutants of neurospora crassa.previous work in other laboratories has shown that several mutagen sensitive mutants of neurospora crassa are extremely sensitive to low levels of histidine in the culture medium. we have shown that wild type neurospora accumulates nicks or breaks in the dna in the presence of histidine. the number of nicks accumulating in histidine sensitive mutants is found to increase in relation to their sensitivity to histidine. although these nicks can be repaired by both wild type and histidine sensitive ...19882969780
ly121019 inhibits neurospora crassa growth and (1-3)-beta-d-glucan synthase. 19882968332
cyclic amp-dependent, constitutive thermotolerance in the adenylate cyclase-deficient cr-1 (crisp) mutant of neurospora crassa.we investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of neurospora crassa. this strain was observed to be much more resistant to a lethal temperature of 50 degrees c than the wild type. this constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic amp (camp, 2.5 mm) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 degrees c, a temperature which fails to induce thermotolerance in the ...19882841035
neurospora mitochondria contain an acyl-carrier protein.mitochondria of neurospora crassa were found to contain a protein which was labelled with [14c]pantothenic acid and which carried an acyl group. this protein, when purified 6000-fold, closely resembled the bacterial and chloroplast acyl-carrier protein(s) [acp(s)] in its physical and chemical properties. the predominant acyl group esterified to the purified protein was 3-hydroxytetradecanoate, as determined by gas chromatographic mass spectrometry. the amino acid sequence of the tryptic peptide ...19883360014
neurospora crassa pyruvate dehydrogenase complex: component characterization, catalytic properties and location of translation.we propose a simplified procedure for the purification of the neurospora crassa pyruvate dehydrogenase complex. the purified complex showed four protein bands with apparent mr values of 53,400, 52,900, 49,000 and 36,900 upon sds-polyacrylamide gel electrophoresis. components, e2 and e3, of n. crassa pyruvate dehydrogenase complex were identified, respectively, as polypeptides 49,000 and 53,400. it can be deduced that component e1 is constituted of two subunits with mr values of 52,900 and 36,900 ...19882965602
preparation of a cell-free translation system from a wild-type strain of neurospora crassa.we describe the preparation of an in vitro translation system from a wild-type strain of neurospora crassa. the system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. the translation products have minimal background and can be clearly analyzed by sds-polyacrylamide gel electrophoresis. the method of preparation of the lysate is simple, fast and reproducible. the procedure should be readily applicable to other filamentous fungi.19882968852
an electrophoretic karyotype of neurospora crassa.a molecular karyotype of neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. the migration of all seven n. crassa chromosomal dnas was defined, and five of the seven molecules were separated from one another. the estimated sizes of these molecules, based on their migration relative to schizosaccharomyces pombe chromosomal dna molecules, are 4 to 12.6 megabases. the seven linkage groups were correlated ...19882967910
molecular cloning and analysis of the regulation of cys-14+, a structural gene of the sulfur regulatory circuit of neurospora crassa.the cys-14+ gene encodes sulfate permease ii, which is primarily expressed in mycelia. cys-14+ is one of a set of sulfur-related structural genes under the control of cys-3+ and scon+, the regulatory genes of the sulfur control circuit. we have cloned cys-14+ from a cosmid library of neurospora crassa dna. a restriction fragment length polymorphism analysis showed that this clone maps to the region of chromosome iv corresponding to the cys-14+ locus. northern blot analyses were used to examine t ...19882898097
cloning of mtr, an amino acid transport gene of neurospora crassa.translocation of neutral aliphatic and aromatic amino acids across the plasma membrane of the ascomycete neurospora crassa requires a functional gene product of the mtr locus. mutations at this locus are defective in transport of those amino acids. we have cloned the mtr+ gene of neurospora crassa from an ordered cosmid library of genomic dna and produced a preliminary restriction map of 2.9 kilobases of genomic dna that encompasses the mtr coding region. we have confirmed that the cloned dna re ...19882843424
secondary structure of the neurospora crassa plasma membrane h+-atpase as estimated by circular a previous communication, a water-soluble, hexameric form of the neurospora crassa plasma membrane h+-atpase was described (chadwick, c. c., goormaghtigh, e., and scarborough, g. a. (1987) arch. biochem. biophys. 252, 348-356). to facilitate physical studies of the hexamers, the h+-atpase isolation procedure has been improved, resulting in a structurally and functionally stable hexamer preparation that contains only 5 to 10% non-atpase protein, approximately 12 mol of enzyme-bound lysophospha ...19882893796
the structural gene for a phosphorus-repressible phosphate permease in neurospora crassa can complement a mutation in positive regulatory gene nuc-1.van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. this was unexpected because the nuc-1 host already contained a resident van+ gene. plasmids carrying van+ complemented a nuc-2 mutation as well. probing of rna from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control ...19882966896
ribosomal dna inheritance and recombination in neurospora crassa.the genetic segregation of ribosomal dna (rdna) in neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (nts) sequences of nine laboratory wild-type strains and wild-collected strains. in an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rdna was shown to be inherited in a simple, stable mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rdna types. ...19882966889
ethanol and carbon-source starvation enhance the accumulation of hsp80 in neurospora neurospora crassa, heat shock results in the induction of 9 to 11 heat shock proteins (hsp), of which hsp80 is the most abundant and the first to be synthesized. the induction of hsp80 was investigated during normal growth (2% sucrose) and under sucrose starvation. transfer of mycelium to a medium supplemented with ethanol stimulated the synthesis of hsp80, even at the normal growth temperature of 28 degrees c. it was also synthesized under carbon starvation conditions, where the medium was s ...19882969770
xanthine dehydrogenase expression in neurospora crassa does not require a functional nit-2 regulatory gene.xanthine dehydrogenase (xdh) is the initial enzyme in the purine catabolic pathway of n. crassa. secondary nitrogen sources such as purines are metabolized when preferred sources of reduced nitrogen (ammonium or glutamine) are unavailable. xdh synthesis is regulated by glutamine repression and uric acid induction. the nit-2 locus is believed to encode a trans-acting positive regulator essential for the expression of genes encoding enzymes involved in secondary pathways of nitrogen acquisition, s ...19882967694
lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase a2: evidence from negative-stain electron microscopy using filipin.the channel protein in the mitochondrial outer membrane of neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase a2. when mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. this suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. it is proposed th ...19882967338
on the role of protein synthesis in the circadian clock of neurospora crassa.inhibitors of protein synthesis reset the biological clocks of many organisms. this has been interpreted to mean either that the synthesis per se of proteins is a step in the oscillatory feedback loop or merely that certain unstable protein(s) are required at certain times of the cycle to complete the feedback loop. we report here that neurospora strains bearing the clock mutation frq-7 are relatively insensitive to the resetting action of the protein-synthesis-inhibitor cycloheximide. protein s ...19882963337
metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of neurospora neurospora crassa, the expression of nit-3, the structural gene which encodes nitrate reductase, is highly regulated and requires both nitrate induction and nitrogen catabolite derepression. the major nitrogen regulatory gene, nit-2, acts in a positive fashion to turn on the expression of nit-3 and other nitrogen-related genes during nitrogen derepression. a second regulatory gene, designated nmr, acts in a negative fashion to repress the expression of nitrate reductase and related enzymes, a ...19882962990
characterization of two allelic forms of neurospora crassa laccase. amino- and carboxyl-terminal processing of a precursor.the complete structures of the laccase genes isolated from two different neurospora crassa wild-type strains are described. the genes were cloned by screening partial genomic dna libraries with a nick-translated laccase-specified 1.36-kilobase sali fragment (germann, u. a., and lerch, k. (1986) proc. natl. acad. sci. u.s.a. 83, 8854-8858) as a hybridization probe. nucleotide sequence analysis revealed the presence of two different allelic forms. they conform to the same structural organization, ...19882961749
regulation of lactate/pyruvate ratios by cyclic amp in neurospora crassa.cyclic amp is thought to have a general role in stimulating the breakdown of carbohydrate reserves and subsequent glycolytic activity. this would be expected to increase the availability of reducing equivalents in the form of cytoplasmic nadh. the current study examines another potential reaction controlling cytoplasmic nadh in the fungus neurospora crassa, that of lactate dehydrogenase, to determine whether it is also regulated by cyclic amp. the cr-1, adenylate cyclase and cyclic amp-deficient ...19882827675
ferricrocin functions as the main intracellular iron-storage compound in mycelia of neurospora crassa.neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome c and some minor unknown compounds. under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferichrome c are predominantly found intracellularly. mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-type n. crassa 74a. the major intracellular targ ...19882978956
defective myo-inositol-1-phosphate synthase production in an inositolless double mutant neurospora crassa strain.a slow growing inl+/- mutant was isolated from an inositol dependent (inl) neurospora crassa strain. the latter strain produces defective myo-inositol-1-phosphate synthase which has residual activity. inositol, similarly to that found in wild and inl mutant strains, represses the enzyme production in the inl+/- strain as well. withdrawing inositol from the medium results in derepression of the enzyme synthesis. derepression is hindered by cycloheximide. inl+/- character in the double mutant is b ...19882977674
mating type response in neurospora crassa. early and transient changes in the patterns of protein synthesis in sexually stimulated mycelia.1. pulse labeling with [35s]-methionine, one-dimensional sds-polyacrylamide gel electrophoresis and fluorography were used to study the pattern of protein synthesis in neurospora crassa mycelia undergoing sexual development. 2. contact of sexually-competent mycelium with cells of the opposite mating type elicited a rapid and transient increase in the synthesis of two predominant proteins of 58 kda and 40 kda localized in the cytosol fraction. 3. marked changes in the pattern of protein synthesis ...19882977101
[mechanism of photoregulation of carotenogenesis in neurospora crassa: the use of mutants]. 19882973407
transcellular ion currents and extension of neurospora crassa hyphae.hyphae of neurospora crassa, like many other tip-growing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. in order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. omission of k+ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. omission of glucose markedly reduced ...19882966862
nitrate assimilation in neurospora crassa: enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures.the nitrate assimilatory pathway in neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. both are alpha 2 type homodimers. enzyme-bound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. one, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. three modes of regulation are imposed on the expression of nitrate assimil ...19882963944
large-scale purification of plasma membrane h+-atpase from a cell wall-less mutant of neurospora crassa. 19882906720
transformation of neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming dna.neurospora trp-1+ transformants, obtained by transforming a trp-1 inl strain with plasmid dna containing the wild type trp1+ gene, were characterized by genetic and southern blot analyses. the transforming trp-1 gene integrated at or near the resident site in all of the trp-1+ transformants obtained with circular dna or dna cut within the trp-1 coding region. the frequency of homologous integration decreased substantially when the donor dna was cleaved outside the trp-1 coding region. the transf ...19882834105
control of nucleotide and erythroascorbic acid pools by cyclic amp in neurospora crassa.udpglucuronic acid and erythroascorbic acid were identified in extracts of the fungus neurospora crassa. the concentrations of these two compounds are estimated, in growing wild type n. crassa, to be about 0.10 and 0.28 mumol/ml of cell water, respectively. the pools of these two compounds are regulated by cyclic amp in neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic amp. the pools of these two compounds are also elevated ...19872825802
rearrangement of duplicated dna in specialized cells of neurospora.introduction of dna into neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, neurospora sequences deleted from the host genome, and foreign sequences. the sequences already represented in the host were rearranged at high frequency in a cross. in general, both elements of the duplication, that from the plasmid and that from the ...19872960455
molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in neurospora crassa.the nit-3 gene of neurospora crassa encodes the enzyme nitrate reductase and is regulated by nitrogen catabolite repression and by specific induction with nitrate. the nit-3 gene was isolated from a cosmid-based genomic library by dual selection for benomyl resistance and for the ability to complement a nit-3 mutant strain using the sibling-selection procedure. the nit-3 gene was subcloned as a 3.8-kilobase dna fragment from a cosmid that carried an approximately 40-kilobase n. crassa dna insert ...19872891138
relationship between two major immunoreactive forms of arginase in neurospora crassa.two major immunoreactive proteins of mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from neurospora crassa. the latter corresponded to the protein used to obtain the antibodies. both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. the relative proportion of the two species was altered in strains containing the nitrogen catabolite regu ...19872890621
circadian rhythms in neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition.the fatty acid compositions of the phospholipids of neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. in liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. by late log phase, fatty acid composition was essentially ...19872889471
antibiotic-induced derepression of the nad-specific glutamate dehydrogenase of neurospora crassa.the catabolic, nad-specific glutamate dehydrogenase (nad-gdh) of neurospora crassa is under carbon catabolite repression. cells grown on a glycolytic carbon source, such as sucrose, have low basal levels of enzyme activity. treatment of repressed cells with either polymyxin b or amphotericin b resulted in derepression of nad-gdh. derepression at the transcriptional level occurred very rapidly (within 30 min) in response to polymyxin b addition but reached a plateau within 2 h. amphotericin b-ind ...19872822659
carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo.the large subunit of carbamoyl phosphate synthase a [carbon-dioxide: l-glutamine amido-ligase (adp-forming, carbamate-phosphorylating), ec] from neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. we have utilized n. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase a to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase a precursor. we found that carboxy ...19872958846
characterization of pi-repressible enzymes secreted in culture media by neurospora crassa wild-type cells and null-type wild-type mycelial cultures of neurospora crassa under pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases i, ii, iii, and iv, 5'-nucleotidase, acid and alkaline nucleases, rnase n1, and a newly detected endonuclease were secreted into the culture media. these enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4. the proteins were examined by polyacrylamide gel electrophoresis in a manner which allow ...19872820943
heat shock induces peroxidase activity in neurospora crassa and confers tolerance toward oxidative stress.heat shock treatment of 14-h-old neurospora crassa mycelium, for 1 h at 48 degrees c, led to the induction of high levels of peroxidase (ec. activity. no significant change was observed in the superoxide dismutase content. colonies formed by plating conidial suspensions on sorbose-medium also exhibited high peroxidase activity following exposure to hyperthermia and were found to be resistant to normally toxic doses of h2o2. thus one of the heat shock proteins of n. crassa has the funct ...19872959286
a eukaryotic repressor protein, the qa-1s gene product of neurospora crassa, is homologous to part of the arom multifunctional enzyme.little is known about the proteins involved in the control of gene expression in eukaryotes. although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. we report here that the qa-1s repressor protein of the fungus neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukary ...19872960822
a novel stimulator of protein phosphorylation in neurospora endogenous thermostable activator of protein kinase iii (pkiii) was purified from 100,000 x g supernatants of neurospora crassa mycelial extracts. this 38,000 dalton polypeptide, clearly separable from calmodulin on p-60 gel filtration, specifically stimulated n. crassa pkiii activity on casein or phosvitin "in vitro" phosphorylation. the factor was only present in the initial growth phase of the fungus. the mechanism of pkiii activation and its possible regulatory role are discussed.19872961977
two developmental stages of neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for the heat shock temperature of 45 degrees c, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of neurospora crassa. both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. at the rna level, however, these two developmental stages responded with different kinetics to elevated temperature. heat shock rnas (for hsp30 and hsp83) accumulated and declined more rapidly in spores t ...19872959857
isolation of telomere dna from neurospora crassa.the most distal known gene on neurospora crassa linkage group vr, his-6, was cloned. a genomic walk resulted in isolation of the telomere at vr. it was obtained from a library in which the endmost nucleotides of the chromosome had not been removed by nuclease treatment before being cloned, and mapping indicates that the entire chromosome end has probably been cloned. sequences homologous to the terminal 2.5 kilobases of dna from vr from these oak ridge n. crassa strains are found at other sites ...19872890097
transcription of neurospora crassa 5 s rrna genes requires a tata box and three internal elements.the sequences required for transcription of neurospora crassa 5s rrna genes have been defined using a comprehensive set of deletion and substitution mutations introduced into two cloned genes. an upstream tata box (consensus tcataga) located at -29 to -24, and three internal regions (d, a and c) localized to +19 to +30, +44 to +57 and +73 to +103, respectively, are absolutely required for transcription in vitro. the tata box fixes the start point of transcription. the a and c regions correspond ...19872960818
partial characterization of gtp-binding proteins in neurospora.six fractions of gtp-binding proteins separated by gel filtration of a mycelial extract containing membrane components of neurospora crassa were partially characterized. [35s]gtp gamma s bound to gtp-binding protein was assayed by repeated treatments with a norit solution and centrifugation. the binding of [35s]gtp gamma s to gtp-binding proteins was competitively prevented in the presence of 0.1 to 1 mm gtp but not in the presence of atp. these gtp-binding proteins fractionated by the gel colum ...19872956953
electron microscopy and image analysis of the mitochondrial outer membrane channel, vdac.the channel protein in the outer membrane of neurospora crassa mitochondria, vdac, forms extended planar crystals on the membrane. the arrays, which are induced by phospholipase a2, are polymorphic, varying from parallelogram (p) to near-rectangular (r) geometry with increased phospholipase treatment. computer-based analysis of projection images of negatively stained vdac arrays indicates that the protein forms a transmembrane channel in the p array. comparison of average images of arrays embedd ...19872442147
a protein required for splicing group i introns in neurospora mitochondria is mitochondrial tyrosyl-trna synthetase or a derivative thereof.the nuclear cyt-18 mutants of neurospora crassa are defective in splicing a number of group i introns in mitochondria. here, cloning and sequencing of the cyt-18 gene show that it contains an open reading frame having significant homology to bacterial tyrosyl-trna synthetases. biochemical and genetic experiments lead to the conclusions that the cyt-18 gene encodes mitochondrial tyrosyl-trna synthetase, that mutations in this gene inhibit splicing directly, and that mitochondrial tyrosyl-trna syn ...19873607872
the influence of copper on the induction of tyrosinase and laccase in neurospora crassa.the influence of copper on the cycloheximide-induced synthesis of the copper-containing enzymes tyrosinase and laccase in neurospora crassa was studied by enzyme activity measurements and immunological means. the amount of active enzyme molecules is far higher when the culture medium is copper-supplemented before cycloheximide induction. the synthesis of the apoproteins is not dependent on the presence of copper. this suggests the existence of a copper-storage protein for which metallothionein i ...19872956123
copper accumulation in the cell-wall-deficient slime variant of neurospora crassa. comparison with a wild-type strain.the copper-uptake process in the cell-wall-deficient slime variant of the fungus neurospora crassa was compared with that in a wild-type strain. in both organisms investigated most of the copper is taken up from the culture medium during the exponential growth period. the wild-type strain, however, accumulates much more copper than does the slime variant. the influence of the copper concentration in the culture medium on the amounts of copper accumulated intracellularly suggests separate ways of ...19872959274
isolation and characterization of non-neuronal enolase (nne) from neurospora crassa and comparison with neuron specific enolase isolated from neuroblastoma cell line ng108.enolase is a vital enzyme of the glycolytic pathway. it exists mainly in two forms, non-neuronal enolase (nne) and neuron specific enolase (nse). neurospora crassa, a filamentous fungus, was used as the source of pure nne, and by using deae-cellulose and a sephadex g-150 column chromatography highly purified enzyme (20.4 fold purification with 54.7 percent recovery) was obtained. the development profile of the enzyme shows a peak value after 90 hours of mycelial growth from conidia of n. crassa. ...19872969242
regulation of carbon and nitrogen flow by glutamate synthase in neurospora crassa.a glycine-resistant neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for nadp-dependent glutamate dehydrogenase (gdh).] this new mutation also conferred resistance to serine and methionine sulphoximine (ms), which are inhibitors of glutamine synthetase (gs). in addition, the mutant obtained grew better on ammonium than the am-132 parental strain. resistance to glycine was not due to increa ...19872959749
molecular cloning and characterization of the cys-3 regulatory gene of neurospora crassa.the regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. the cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. the library used (pral1) contained inserts of sau3a partial digest fragments of about 9 kilobases as well as the neurospora qa-2+ gene. double selection ...19872886908
deficiency in mrna splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.molecular cloning and characterization of cytochrome c cdna clones of neurospora crassa wild-type (74a) and a cytochrome c-deficient mutant (cyc1-1) are described. southern blot analysis of genomic dna indicates that only one cytochrome c gene exists in the n. crassa genome. the cdna sequence of the wild-type cytochrome c confirmed the previously determined protein sequence. sequence analysis of the cyc1-1 cdna for cytochrome c revealed the presence of a larger open reading frame, owing to the p ...19872820723
differential dna methylation during the vegetative life cycle of neurospora crassa.isotope dilution gas chromatography-mass spectrometry analysis of genomic dnas isolated from the different growth phases of neurospora crassa revealed significant differences in the amounts of 5-methylcytosine; the mol % of 5-methylcytosine was 0.36 in conidia (asexual spores), 0.40 in conidial germlings cultured for 3 h, 0.24 in mycelial cells that had grown exponentially for 6 and 12 h, and 0.40 in stationary-phase mycelial cells. these results indicate an approximate inverse correlation betwe ...19872953709
arginine-specific carbamoyl phosphate metabolism in mitochondria of neurospora crassa. channeling and control by arginine.citrulline is synthesized in mitochondria of neurospora crassa from ornithine and carbamoyl phosphate. in mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. we have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. very little of this carbamoyl phosphate esca ...19872953716
characterization of nit-2, the major nitrogen regulatory gene of neurospora crassa.the nit-2 gene is the major nitrogen-regulatory gene of neurospora crassa, and under conditions of nitrogen limitations, it turns on the expression of various unlinked structural genes which specify nitrogen-catabolic enzymes. the nit-2 gene was subcloned as a 6-kilobase (kb) dna fragment from a cosmid that carried approximately a 40-kb n. crassa dna insert. the nit-2 gene was localized in a dna segment of approximately 3.5 kb and was shown to correspond to a unique dna sequence located on linka ...19872885741
a single precursor protein for two separable mitochondrial enzymes in neurospora crassa.the arg-6 locus of neurospora crassa encodes two early enzymes of the arginine biosynthetic pathway, acetylglutamate kinase and acetylglutamyl-phosphate reductase. previous genetic and biochemical analyses of this locus and its products showed that: 1) strains carrying polar nonsense mutations in the acetylglutamate kinase gene lacked both enzyme activities (davis, r.h., and weiss, r.l. (1983) mol. gen. genet. 192, 46-50), and 2) the proteins isolated from mitochondria were completely separable ...19873032945
gmp-stimulation of the cyanide-insensitive mitochondrial respiration in heat-shocked conidia of neurospora mitochondria of heat-shocked conidia of neurospora exogenous nadh and succinate were oxidized mainly via the alternative, hydroxamate-sensitive pathway (70%) and only 30% via the cytochromic, cyanide-sensitive pathway which was predominant in untreated conidia; the alternative oxidase pathway was markedly stimulated by guanosine 5'-monophosphate (gmp).19873032673
location of a dicyclohexylcarbodiimide-reactive glutamate residue in the neurospora crassa plasma membrane h+-atpase.the proton pump (h+-atpase) found in the plasma membrane of the fungus neurospora crassa is inactivated by dicyclohexylcarbodiimide (dccd). kinetic and labeling experiments have suggested that inactivation at 0 degrees c results from the covalent attachment of dccd to a single site in the mr = 100,000 catalytic subunit (sussman, m. r., and slayman, c. w. (1983) j. biol. chem. 258, 1839-1843). in the present study, when [14c]dccd-labeled enzyme was treated with the cleavage reagent, n-bromosuccin ...19872881924
induced pelletized growth of neurospora crassa for tyrosinase biosynthesis in airlift fermenters. 198718576515
some aspects of the regulation of pyruvate kinase levels in neurospora crassa.pyruvate kinase levels were monitored in neurospora crassa mycelium (grown on different carbon sources for varying time intervals) by immunoprecipitation using polyclonal antibodies raised against a purified enzyme preparation. pyruvate kinase specific mrna was demonstrated by hybridization of northern and dot blots of total rna with a n. crassa pyruvate kinase gene fragment. two pyruvate kinase specific mrna species were detected in mycelia of all ages examined. an age-dependent and carbon sour ...19873036325
import of cytochrome c into mitochondria. cytochrome c heme lyase.the import of cytochrome c into mitochondria can be resolved into a number of discrete steps. here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from neurospora crassa. a new method was developed to measure directly the linkage of heme to apocytochrome c. this method is independent of conformational changes in the protein accompanying heme attachment. tryptic peptides of [35s]cysteine-labelled apocytochrome c, and of enzymat ...19873030750
isolation and characterization of coated vesicles from filamentous fungi.coated vesicles have been shown to exist in neurospora crassa (ascomycetes) and uromyces phaseoli (basidiomycetes) growing germlings. separation of coated vesicles in both fungi was obtained when the high-speed (100,000g) pellet was fractioned on a sephacryl s-1000 gel filtration column, according to the procedure of mueller and branton. electron micrographs of negatively stained coated vesicles from fractions of gel filtration show the same striking lattice coated vesicles similar to vertebrate ...19872885195
circadian rhythms in neurospora crassa: membrane composition of a mutant defective in temperature compensation.the cel mutant of neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees c, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. during growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. the lev ...19872950925
three-dimensional structure of nadh: ubiquinone reductase (complex i) from neurospora mitochondria determined by electron microscopy of membrane crystals.nadh: ubiquinone reductase (electron transfer complex i) has been isolated from neurospora crassa mitochondria as a monodisperse protein-phospholipid-triton x-100 complex (1:0.04:0.15, by weight). the enzyme is in the monomeric state, has a protein molecular weight of 610,000 and consists of about 25 different subunits. membrane crystals of the enzyme complex have been prepared by adding mixed phospholipid-triton x-100 micelles and then removing the triton by dialysis. diffraction patterns of th ...19872956429
h-atpase activity from storage tissue of beta vulgaris: iv. n,n'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane h-atpase.the molecular weight and isoelectric point of the plasma membrane h(+)-atpase from red beet storage tissue were determined using n,n'-dicyclohexylcarbodiimide (dccd) and a h(+)-atpase antibody. when plasma membrane vesicles were incubated with 20 micromolar [(14)c]-dccd at 0 degrees c, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. a close correlation between [(14)c]dccd labeling of the 97,000 dalton protein and the extent of atpase ...198716665290
characterization of two abundantly expressed constitutive genes of neurospora crassa.two abundantly expressed, constitutive genes of neurospora crassa were isolated during differential screening of neurospora genomic libraries. the coding regions of these two genes, designated rlf1 and rlf3, were identified by hybridization of the cloned dna sequences with cdna probes made from polyadenylated rna. the rlf3 gene was carried on a 15-kilobase neurospora bamhi dna fragment present in a lambda 1059 recombinant; a 2-kilobase restriction fragment that contains rlf3 was subcloned into p ...19873032385
expression of qa-1f activator protein: identification of upstream binding sites in the qa gene cluster and localization of the dna-binding domain.the qa-1f regulatory gene of neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. this activator protein was expressed in insect cell culture with a baculovirus expression vector. the activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. one site is located between the divergently transcribed qa-1f and qa-1s regulatory genes, corroborating prior ev ...19872951591
nonsense mutations of the ornithine decarboxylase structural gene of neurospora crassa.ornithine decarboxylase (odc) (ec is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. some have speculated that the enzyme protein may have a role in the synthesis of rrna in addition to its role in catalyzing the decarboxylation of ornithine (g. d. kuehn and v. j. atmar, fed. proc. 41:3078-3083, 1982; d. h. russell, proc. natl. acad. sci. usa 80:1318-1321, 1983). to test this possibility, we sought mu ...19872951589
signal for dna methylation associated with tandem duplication in neurospora crassa.most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of neurospora crassa. the region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5s rrna gene. the repeated elements have diverged about 15% by the occurrence of numerous cg to ta mutations, which probably resulted from deamination of methylated cytosines. most but not all common laboratory strains of n. crassa have methylated duplicated dna at the zeta-eta locus. however, many ...19872951588
two n-hydroxylaminopurines are highly mutagenic in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of neurospora crassa.3 purine analogs were tested for their mutagenic activities in the ad-3 forward-mutation test in heterokaryon 12 (h-12) of neurospora crassa. in growing cultures of h-12, the n-hydroxylaminopurines 2-amino-6-n-hydroxylaminopurine (aha) and 6-n-hydroxylaminopurine (hap) are potent and strong mutagens, respectively, whereas 2-aminopurine (ap) is a weak mutagen. aha and hap are about equally mutagenic at low doses, but aha is more mutagenic than hap at high doses. despite their potent mutagenicity ...19872950320
metabolic utilization of 57fe-labeled coprogen in neurospora crassa. an in vivo mössbauer study.mössbauer spectra of whole cells of neurospora crassa arg-5 ota aga (a siderophore-free mutant) show that the siderophore coprogen is accumulated inside the cell as an entity. 57fe from 57fe-labeled coprogen is slowly removed from the complex (45% in 27 h). the rate of removal depends on the degree of iron starvation of the cells. the distribution of 55fe from [55fe]coprogen in vacuoles, membranes, and cytoplasm has been also determined. from this it is clear that coprogen is accumulated in the ...19872951253
role of nitrogen in the photoinduction of protoperithecia and carotenoids in neurospora crassa.nitrogen, as kno3 or nh4no3, can inhibit the photoinduction of protoperithecia in neurospora crassa when present in the medium at a high concentration but does not inhibit the photoinduction of carotenoids. the point at which the presence of high nitrogen levels is no longer inhibitory is 5 h after illumination.198724232879
intracellular and extracellular cyclic nucleotides in wild-type and white collar mutant strains of neurospora crassa: temperature dependent efflux of cyclic amp from mycelia.cyclic amp and cyclic gmp were released into the growth medium of mycelia of neurospora crassa wild-type strains st.l.74a and em5297a and by white collar-1 and white collar-2 mutant strains. after growth for 6 days at 18 degrees c, there were 2.19 (st.l.74a), 5.83 (em5297a), 1.38 (white collar-1), and 1.10 (white collar-2) nanomoles of cyclic amp per gram dry weight of mycelia in the growth medium. these values corresponded to concentrations of cyclic amp of between approximately 10 and 50 nanom ...198716665253
three class i introns in the nd4l/nd5 transcriptional unit of neurospora crassa mitochondria.the overlapping nd4l and nd5 genes of neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. all three intervening sequences are class i introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. they contain long open reading frames (orfs; from 306 to 425 codons long) that are continuous and in frame with th ...19872953954
a hexameric form of the neurospora crassa plasma membrane isolated by our recently developed large-scale procedure, the neurospora plasma membrane h+-atpase exists as a homogeneous, oligomeric complex of 105,000-da monomers with a molecular mass equivalent to a spherical protein of about 1 million da, as judged by its behavior during chromatography on calibrated columns of sepharose cl-6b and cl-4b. treatment of this complex with the nonionic detergent, tween 20, followed by sepharose column chromatography in the presence of this detergent produces ...19872880563
nuclear endo-exonuclease of neurospora crassa. evidence for a role in dna repair.the major nuclease activity in nuclei of mycelia of neurospora crassa has been identified as that of endoexonuclease, an enzyme purified and characterized previously from mitochondria and vacuoles which acts endonucleolytically on single-stranded dna and rna and possesses highly processive exonuclease activity with double-stranded dna. cross-contamination from the other organelles was eliminated as a source of the activity. endo-exonuclease of nucleoplasm, chromatin, and nuclear matrix showed 80 ...19873025215
isolation and characterization of a dna-uptake-stimulating protein from the culture medium of neurospora crassa slime strain.a protein fraction was purified to homogeneity from the culture medium of the wall-less (slime) strain of neurospora crassa (fgsc 1118), which proved to be identical with dna-uptake-stimulating factor (designated dusf), which has been described earlier [schablik, m. and szabó, g. (1981) fems microbiol. lett. 10, 395-397]. the quantity of dusf is measured by the amount of [3h]dna uptake by neurospora cells at standard conditions. its relative molecular mass was 230,000. it has an isoelectric poin ...19872949969
monophenol monooxygenase from neurospora crassa. 19873037257
applicability of the equations of freundlich and langmuir to the adsorption of the azo dye procion scarlet on paramorphic colonies of neurospora crassa.experiments on the adsorption of procion scarlet mx-g by normal hyphae and by paramorphic colonies of neurospora crassa were performed at ph 2.5, 4.5 and 6.5 at 30 degrees c. the measured adsorption isotherms were evaluated by the freundlich and langmuir equations. the removal of dye was most effective at ph 2.5 and more dye was adsorbed per unit mass of cells in the paramorphic cultures than in the normal hyphae. the statistical tests showed langmuir's equation to give a better fit to the adsor ...19872968127
possible link between circadian rhythm and heat shock response in neurospora crassa.3-h pulses of elevated temperatures (30 degrees c, 35 degrees c, 40 degrees c) phase shift the circadian conidiation rhythm of neurospora crassa. the phase and amplitude of the phase response curves (prc) were measured in wild type (frq+) and frequency mutants (frq 1, frq 7). the dose dependence of the phase shifts was compared to the dose dependence of total protein synthesis inhibition and heat shock protein induction in the three strains. all processes showed an almost linear dependence on te ...19872963703
regulation of the qa gene cluster of neurospora crassa. 19872961304
differential synthesis and replication of dna in the neurospora crassa slime mutant versus normal cells: role of carcinogens.small quantities of carcinogens, dl-ethionine, thiotepa, actinomycin d, and 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (ccnu) stimulated in vitro deoxyribonucleic acid (dna) synthesis of the slime mutant of neurospora crassa, while there was practically no effect on the dna from the normal wild type 74a strain. all of these compounds caused increased strand separation in the mutant dna of n. crassa, but no separation of normal dna strands. the growth (in vivo tests) of the n. crassa slime muta ...19872959890
isolation and regulation of expression of the neurospora crassa copper metallothionein gene.the n. crassa cumt gene has been cloned and its nucleotide sequence determined. to this end an mt specific undecanucleotide was synthesized and used for cdna synthesis with enriched mt mrna as a template. sequence analysis of the cdna obtained allowed the synthesis of a unique 21mer which was used as a hybridization probe to screen a genomic dna library of n. crassa. several positive clones were isolated and subjected to restriction and sequence analysis. in agreement with the published amino ac ...19872959528
isolation and characterization of an extracellular lipase from the conidia of neurospora crassa.a triacylglycerol lipase (ec from the conidia of neurospora crassa was purified and characterized. the enzyme was purified by sephadex g-100 column chromatography. homogeneity was checked by page, and isoelectric focusing gave a single band corresponding to a pi of 6.4. the enzyme had an apparent mr 54000 +/- 1000 as determined by gel filtration. sds-page gave a single band of mr 27000, suggesting the presence of two identical subunits. this lipase preferred triglycerides with c16- and ...19872958597
the arom multifunctional enzyme from neurospora crassa. 19872955200
analysis of mutational lesions of acetate metabolism in neurospora crassa by 13c nuclear magnetic resonance.the adaptation of neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13c nuclear magnetic resonance. extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13c]acetate as the sole carbon source. the label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13c-enriched trehalose was evident, indicating that gluconeoge ...19872947898
primary structure of the neurospora crassa small subunit ribosomal rna coding region. 19862948156
ascus development in two temperature-sensitive four-spore mutants of neurospora crassa.two nonallelic four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. expression depends on temperature. the four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees c) for fsp-1 or to low temperatures (15-20 degrees c) for fsp-2. heterozygous fsp-1 x fsp-1+ crosses make eight-spored asci at 15-20 degrees c but produce many four- ...19862950989
a recessive circadian clock mutation at the frq locus of neurospora crassa.a circadian clock mutant of neurospora crassa, the most distinctive characteristic of which is the complete loss of temperature compensation of its period length, maps to the frq locus where seven other clock mutants have previously been mapped. this mutant, designated frq-9, is recessive to the wild-type allele and to each of the other frq mutants; thus, it differs from the other mutants, which show incomplete dominance to wild type and to each other. complementation analysis suggests either th ...19862948874
reversal of a neurospora translocation by crossing over involving displaced rdna, and methylation of the rdna segments that result from translocation oy321 of neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group i. in crosses of translocation x translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to normal. genetic, cytological, and molecular evidence indicates that reversion is the resul ...19862947829
pyrophosphate-caused inhibition of the aminoacylation of trna by the leucyl-trna synthetase from neurospora crassa.inorganic pyrophosphate inhibits the aminoacylation of trnaleu by the leucyl-trna synthetase from neurospora crassa giving very low kapp.i, ppi values of 3-20 microm. the inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, atp and trna are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. a kinetic analysis of this mechanism shows that the measured kapp.i values do not give the real ...19863021454
an upstream signal is required for in vitro transcription of neurospora 5s rna genes.the dna sequences upstream of the 5s rna genes in neurospora crassa are largely different from one another, but share a short consensus sequence located in the segment 29 to 26 nucleotides preceding the transcribed region. differences among flanking sequences do not appear to affect transcription. deletion analysis indicates, however, that a dna segment including the conserved "tata box" is required for in vitro transcription of neurospora 5s rna genes.19863025558
purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of neurospora crassa mitochondria.a simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of neurospora crassa mitochondria is described. after fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. covalent chromatography on thiol-activated sepharose cl-4b allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2- ...20152947635
amino acid sequence of the plasma membrane atpase of neurospora crassa: deduction from genomic and cdna sequences.the plasma membrane of neurospora crassa contains an electrogenic h+-atpase (ec, for which we have isolated and sequenced both genomic and cdna clones. the atpase gene is interrupted by four small introns (58-124 base pairs). it encodes a protein of 920 amino acids (mr, 99,886) possessing as many as eight transmembrane segments. the neurospora atpase shows significant amino acid sequence homology with the na+,k+- and ca2+-transporting atpases of animal cells, particularly in regions th ...19862876429
dnase i hypersensitive sites within the inducible qa gene cluster of neurospora crassa.dnase i hypersensitive regions were mapped within the 17.3-kilobase qa (quinic acid) gene cluster of neurospora crassa. the 5'-flanking regions of the five qa structural genes and the two qa regulatory genes each contain dnase i hypersensitive sites under noninducing conditions and generally exhibit increases in dnase i cleavage upon induction of transcription with quinic acid. the two large intergenic regions of the qa gene cluster appear to be similarly organized with respect to the positions ...19862944110
genetic and biochemical identification of the glutamate synthase structural gene in neurospora crassa.neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions. despite the physiological importance of glutamate synthase, little is known about the genetics of its expression. to identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. all mutations are recessive to the wild-type allele and belong to the same complementation group as the previously described en(am)-2 (c24) mutation. two lines of evidence in ...19862943726
molecular recognition of siderophores in fungi: role of iron-surrounding n-acyl residues and the peptide backbone during membrane transport in neurospora crassa.recognition of ferric siderophores in neurospora crassa was found to depend on the number and kind of n-acyl residues that surrounded the iron coordination center. in the coprogen series, uptake decreased in the order of coprogen, neocoprogen i, and neocoprogen ii, indicating that gradual replacement of the n-transanhydromevalonyl groups by n-acetyl groups had an adverse effect on uptake. the reverse effect was observed in the ferrichrome series, where uptake decreased in the order of ferrichrys ...19862943724
characterization of an essential arginine residue in the plasma membrane h+-atpase of neurospora crassa.treatment of the plasma membrane h+-atpase of neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees c, ph 7.0, leads to a marked inhibition of atpase activity. mgatp, the physiological substrate of the enzyme, protects against inactivation. mgadp, a competitive inhibitor of atpase activity with a measured ki of 0.11 mm, also protects, yielding calculated kd values of 0.125 and 0.115 mm in the presence of phenylglyoxal and 2,3-butanedione, respective ...19862874143
control of arginine metabolism in neurospora crassa. role of feedback inhibition.flux through the arginine biosynthetic pathway of neurospora crassa was measured under a variety of physiological conditions. flux persisted, although at a reduced rate, in mutant strains resistant to feedback inhibition even after prolonged growth in the presence of exogenous arginine. flux reverted to the uninhibited rate more quickly in feedback-resistant strains than in wild type strains upon removal of exogenous arginine. these results rule out enzyme repression as a major factor in control ...19862942539
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