TitleAbstractYear(sorted descending)
identification of the major cytoplasmic regions of the neurospora crassa plasma membrane h(+)-atpase using protein chemical techniques.the transmembrane topography of the neurospora crassa plasma membrane h(+)-atpase has been investigated using purified, reconstituted components and direct protein chemical techniques. reconstituted proteoliposomes containing h(+)-atpase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the h(+)-atpase as recently described (hennessey, j.p., jr., and scarborough, g. (1990) j. biol. c ...19902144525
nucleotide sequence and dna recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of neurospora crassa.the nitrogen regulatory circuit of neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. this set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. the complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. the alc gene ...19902148685
gene sequence and analysis of hsp30, a small heat shock protein of neurospora crassa which associates with mitochondria.hsp30 is a small heat shock protein of neurospora crassa which earlier studies suggested may associate with mitochondria during cellular heat shock. we show here that the association of hsp30 with mitochondria is reversible and that hsp30 dissociates after cells are returned to normal temperature. we sequenced the gene for hsp30 and defined its transcript by s1 nuclease analysis and cdna sequencing. the gene apparently is present in the genome as a single copy, and it contains no introns. the en ...19902144284
sorting pathways of mitochondrial inner membrane proteins.two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. in the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. they become proteolytically processed, interact with the 60-kda heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. the sorting of subunit 9 of neurosp ...19902145157
restricted activation of general amino acid control under conditions of glutamine limitation in neurospora neurospora crassa limitation for single amino acids normally results in increased formation of enzymes required for amino acid synthesis via 'general amino acid control'. glutamine limitation, however, led to comparatively low and delayed derepression of enzyme synthesis. nitrate reductase activity increased steeply under these conditions confirming that de novo protein synthesis could occur. derepression levels were unaffected by addition of glutamine-derived metabolites. only small and dela ...19902148607
glutamine metabolism and cycling in neurospora crassa.evidence for the existence of a glutamine cycle in neurospora crassa is reviewed. through this cycle glutamine is converted into glutamate by glutamate synthase and catabolized by the glutamine transaminase-omega-amidase pathway, the products of which (2-oxoglutarate and ammonium) are the substrates for glutamate dehydrogenase-nadph, which synthesizes glutamate. in the final step ammonium is assimilated into glutamine by the action of a glutamine synthetase (gs), which is formed by two distinct ...19902145504
behavior of the [mi-3] mutation and conversion of polymorphic mtdna markers in heterokaryons of neurospora crassa.we have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtdna markers in heterokaryotic cultures of neurospora crassa. previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of ecori-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. we have confirmed this observation and determined by dna sequence analysis that the insertion in the ecori-5 fragment corresponds precisely to an optional intron that ...19901977658
isolation and characterization of a neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase.we report the isolation and characterization of a neurospora crassa glutamine synthetase (gs) mutant altered in one of the two polypeptides (gs alpha) of this enzyme. we used the gln-1br8 mutant strain that synthesizes only the gs alpha monomer and lacks the gs beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-dl-methionine-sr-sulfoximine (alpha-me-mso), an inhibitor of gs activity. the gs activity of the gln-1br8;alpha-me-msor strain drastically reduced its ...19901975579
nucleotide sequence of a full-length cdna coding for the mitochondrial precursor protein of the beta-subunit of f1-atpase from neurospora crassa. 19902144339
effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes: a study on mycelial and wall-less phenotypes of the fz; sg; os-1 ('slime') triple mutant of neurospora crassa.the production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain rcp-3) of a cross 'slime' x wild type. the unrelated stable 'slime' strain fgsc 1118 was also studied. the synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-cat ...19902262786
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. vi. induction kinetics of gene/point mutations, multilocus deletions and multiple-locus mutations.genetic fine-structure analysis of x-ray-induced specific-locus mutants in the ad-3 region of two-component heterokaryons of neurospora crassa has shown that gene/point mutations, multilocus deletions and multiple-locus mutations are induced. when the dose-response curves for these classes of ad-3 mutants were plotted, it was demonstrated that x-ray-induced gene/point mutations (ad-3r) increased linearly with x-ray dose and x-ray-induced multilocus deletions increased as the square of the x-ray ...19902143556
biochemical, genetic and ultrastructural defects in a mitochondrial mutant (er-3) of neurospora crassa with senescence phenotype.the structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant er-3 of neurospora crassa are described. the mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. however, the total whole cell respiration rate of the mutant was 33% gre ...19902169558
analysis of conventional and in vitro generated mutants of nmr, the negatively acting nitrogen regulatory gene of neurospora crassa.the nmr gene is the major negative regulatory gene in the nitrogen control circuit of neurospora crassa, which, together with positive regulatory genes, governs the expression of multiple unlinked structural genes of the circuit. possible functional domains of the nmr protein were investigated by mutational analyses using three different approaches. first, the polymerase chain reaction was used to clone the nmr locus from two conventional mutants, v2m304 and ms5, and the mutant amino acid codons ...19902148799
distant upstream regulatory sequences control the level of expression of the am (gdh) locus of neurospora crassa.we have constructed deletions in the 5' noncoding sequences of the cloned neurospora crassa am gene. vectors with a truncated fragment of the am gene were used in transformation experiments to introduce the deletions into the chromosome by homologous recombination. analysis of glutamate dehydrogenase (gdh) expression by enzyme assay and immunoblots, as well as northern and dot blots of poly (a)+ rna, in the deletion strains indicates that there are two upstream regulatory sequences that control ...19902147126
the cellulase complex of neurospora crassa: activity, stability and release.the temperature and ph optima, and the temperature and ph stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus neurospora crassa were investigated. the effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of cellulase complex were also examined. for the different enzymes of the complex, activity maxima occurred between ph 4.0 and 7.0, with ph 5.0 being close to optimal for stability of all. temperature optim ...19902146364
assembly of nadh: ubiquinone reductase (complex i) in neurospora mitochondria. independent pathways of nuclear-encoded and mitochondrially encoded subunits.nadh:ubiquinone reductase, the respiratory chain complex i of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one fmn, probably one internal ubiquinone and at least four iron-sulphur clusters. we are studying the assembly of the enzyme in neurospora crassa. the flux of radioactivity in cells that were pulse-labelled with [35s]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. la ...19902141652
a polypeptide of 59 kda is associated with bundles of cytoplasmic filaments in neurospora crassa.complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus neurospora crassa. they were enriched by differential centrifugation and purified by permeation chromatography. the filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. the main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kda (p59nc), which represents 4-5% of the total n. crassa protein ...19902141976
intracellular sodium content of a wall-less strain of neurospora crassa and effects of insulin: a 23na-nmr study.23na-nmr has been used to investigate some factors influencing the sodium content of a wall-less strains of neurospora crassa. the shift reagent tm(dotp)h2(nh4)3 proved useful for this purpose, while several other reagents, previously used by others, were found to be unsuitable for use with these cells. when the cells were grown, washed and resuspended in medium containing sodium (25.3 mm), the intracellular sodium concentration was calculated to be 11.9 +/- 1.4 mm. this value rose within two mi ...19902142438
processing of precursor proteins by plant mitochondria.precursor proteins from neurospora crassa were correctly processed by a matrix extract from vicia faba and cauliflower mitochondria. processing yielded mature protein of the same molecular mass as mature neurospora protein. the processing activity has two components. one is antigenically related to and of the same molecular mass as the processing enhancing protein of neurospora. the second component was not recognized by antibody to the matrix processing protease from neurospora mitochondria. th ...19902140932
duplication-induced mutation of a new neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.a cloned neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic dna and the normally located gene. such changes are typical of the duplication-induced premeiotic disruption (the rip effect) first described by selker et al. (e. u. sel ...19902140429
molecular cloning and characterization of alc the gene encoding allantoicase of neurospora crassa.purines can be utilized as a secondary nitrogen source by neurospora crassa during conditions of nitrogen limitation. the expression of purine catabolic enzymes is governed by the nitrogen regulatory circuit and requires induction by uric acid. the major positive-acting nitrogen regulatory gene, nit-2, turns on the expression of the purine catabolic enzymes, which may also be subject to negative regulation by a second control gene, nmr. we have cloned alc, the structural gene which encodes allan ...19901978237
chemical state of the cysteine residues in the neurospora crassa plasma membrane h(+)-atpase.the plasma membrane h(+)-atpase of neurospora crassa was treated with 5,5'-dithiobis(2-nitrobenzoate) to determine its cysteine content and with 2-nitro-5-thiosulfobenzoate to determine its cystine content. six and seven mol of thiols/mol of h(+)-atpase were detected in the 5,5'-dithiobis(2-nitrobenzoate) and 2-nitro-5-thiosulfobenzoate reactions, respectively, indicating that 6 of the 8 cysteine residues in the molecule are present as free cysteines and that 2 are present in disulfide linkage. ...19902139659
blue light photoreception in neurospora circadian rhythm: evidence for involvement of the flavin triplet state.the mechanism of the photoreceptor acting on the circadian conidiation rhythm of neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) phase shifts were not abolished by removal of oxygen prior to illumination. (3) oxygen inhibited phase shifts when introduced into the cultures after light treatment. it is proposed that the blue light photoreceptor for the circadian clock of neurospora crassa acts (1 ...19902367558
development of thermotolerance in neurospora crassa by heat shock and other stresses eliciting peroxidase induction.hyperthermia, cdcl2, sodium arsenite, and h2o2 led to the rapid appearance of high levels of peroxidase in neurospora crassa cultures and induced tolerance toward normally lethal temperatures in 60-h-old colonies. intracellular superoxide dismutase levels did not correlate with the development of thermotolerance.19902139653
the genetics of polyamine synthesis in neurospora mutations of the polyamine pathway of neurospora crassa fell into three categories. the majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. one mutation, jp100, defining the new spe-2 locus, eliminated s-adenosyl-methionine decarboxylase and led to putrescine accumulation. revertants of this mutation suggested that the locus encodes the enzyme. two other mutations, lv105 and jp120, defined a third locus, spe-3. strains with these mutations also accumulate ...19902139316
characterization of telomere dna from neurospora crassa.the nucleotide sequence of the telomere at the right end of linkage group v (vr) in the standard or23-iv-a strain of the filamentous fungus, neurospora crassa, reveals the following features. at the chromosome terminus, tandem repeats of the hexanucleotide ttaggg are present. immediately centromere-proximal to the simple sequence repeat is a more complex element called pogo that is reiterated 5-10 times in the genomes of various neurospora strains. the element possesses several features characte ...19901971801
light-induced dephosphorylation of a 33 kda protein in the wild-type strain of neurospora crassa: the regulatory mutants wc-1 and wc-2 are abnormal.light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of neurospora crassa. the regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. because the wc genes have previously been implicated in photodifferentiation (f. degli innocenti and v. e. a. russo, genetic analysis of blue light-induced responses in neurospora crassa, in h. senger (ed.), blue light effects in biological systems, springer- ...19902140412
very low atp/adp ratios with aging of the natural death senescence mutant of neurospora crassa.the natural death (nd) mutant of the fungus neurospora crassa, unlike the wild-type, undergoes an aging process, which leads to the cessation of growth. it is shown here that the atp/adp ratio of the mutant declines with age to about 3:1 whereas other strains of neurospora in the same growth medium maintain ratios of about 8 to 9:1. the decline in atp/adp ratio is not caused by the cessation of growth of the mutant. the results suggest, rather, that the cessation of growth may be caused, in part ...19902139154
relationship of vector insert size to homologous integration during transformation of neurospora crassa with the cloned am (gdh) gene.we used lambda and plasmid vectors containing the am+ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. a total of 199 transformants were examined with the potential to yield am+ transformants by homologous recombination. when we used vectors that had 9.1 kb of homology with the chromosomal dna, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a pl ...19902157957
dna methylation and chromatin structure: a view from understanding of the function and control of dna methylation in eukaryotes has been elusive. studies of neurospora crassa have led to a model that accounts for the chromosomal distribution of methylation and suggests a basic function for dna methylation in eukaryotes.19902139257
determination of the inactivating alterations in two mutant alleles of the neurospora crassa cross-pathway control gene cpc-1.cpc-1 is the locus specifying what is believed to be the major trans-activating transcription factor that regulates expression of amino acid biosynthetic genes subject to cross-pathway control in neurospora crassa. mutants altered at this locus are incapable of the global increase in gene expression normally seen in response to amino acid starvation. using polymerase chain reaction methodology we have cloned and sequenced the inactive mutant allele, cpc-1 (cd15). the cpc-1 (cd15) mutation was fo ...19902138111
synaptic adjustment of inversion loops in neurospora crassa.heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. this synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cm long loops.19902138110
nit-2, the major nitrogen regulatory gene of neurospora crassa, encodes a protein with a putative zinc finger dna-binding domain.the nitrogen regulatory circuit of neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. the positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. the complete nucleotide sequence of the nit-2 gene was determined. the nit-2 mrna is 4.3 kilobases long and has a long ...19902137552
cis,cis-cyclohexane 1,3,5-triol polyphosphates release calcium from neurospora crassa via an unspecific ins 1,4,5-p3 receptor.we investigated the effects of new inositol 1,4,5-trisphosphate analogues on the release of ca2+ from isolated vacuoles of neurospora crassa. tri-o-butyryl-inositol 1,4,5-trisphosphate and a set of cis,cis-cyclohexane 1,3,5-triol bis-(cht-p2) and trisphosphates (cht-p3) gave an increase in free ca2+ as measured directly with fura-2, a ca2(+)-chelator. however, inositol 1,4-bisphosphate, 6-o-palmitoyl-inositol 4,5-bisphosphate and trans-cyclohexane 1,2-diol bisphosphate (trans chd-p2) did not ind ...19902154977
the range of amino acids whose limitation activates general amino-acid control in neurospora crassa.several amino-acid synthetic enzymes, belonging to arginine, glutamine, leucine, lysine and phenylalanine biosynthesis, respectively, were investigated under conditions of reduced availability of any one of 16 out of the 20 amino acids represented in proteins. the enzymes showed simultaneous derepression under each condition, albeit to different degrees. derepression was abolished and the remaining basal enzyme levels reduced by mutations at the cpc-1 locus which governs general amino-acid contr ...19902138581
cellulase production by neurospora crassa: purification and characterization of cellulolytic studies on cellulase production by the cell-1 mutant of neurospora crassa, eight enzymes (three exoglucanases, four endoglucanases, and one beta-glucosidase) were identified and characterized by gel filtration, ion exchange chromatography, and chromatofocusing. after purification, each of the proteins ran as a single band in polyacrylamide gel electrophoresis, using both native and denaturing gels. the molecular weights of the proteins were found to be between 70,000 and 22,000 daltons, and a ...19901368541
de novo fatty acid synthesis mediated by acyl-carrier protein in neurospora crassa mitochondria.the acyl-carrier protein (acp) in neurospora crassa mitochondria [brody, s. & mikolajczyk, s. (1988) eur. j. biochem. 173, 353-359] mediated a cerulenin-sensitive, de novo fatty acid synthesis independent of the fatty acid synthetase complex present in the cytoplasm. incubation of mitochondria with [2-14c]malonate labeled only the acp as indicated by autoradiography after sds/page. under these in vitro conditions atp was required for the initial acyl-acp formation, but further elongation require ...19902137086
nucleotide sequence of the gene coding for cyclophilin/peptidyl-prolyl cis-trans isomerase of neurospora crassa. 19902137907
direct evidence for the cytoplasmic location of the nh2- and cooh-terminal ends of the neurospora crassa plasma membrane h+-atpase.reconstituted proteoliposomes containing neurospora plasma membrane h+-atpase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the nh2 and cooh termini of the h+-atpase relative to the lipid bilayer. treatment of the proteoliposomes with trypsin in the presence of the h+-atpase ligands mg2+, atp, and vanadate produces approximately 97-, 95-, and 88-kda truncated forms of the h+-atpase similar to those already known to resu ...19902136741
on the role of ca2(+)-calmodulin-dependent and camp-dependent protein phosphorylation in the circadian rhythm of neurospora crassa.pulses of some ca2+ channel blockers (dantrolene, co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5-9 h) phase shifts of the circadian conidiation rhythm of neurospora crassa. pulses of high ca2+, or of low ca2+, a ca2+ ionophore (a23187) together with ca2+, and other ca2+ channel blockers (la3+, diltiazem), however, caused only minor phase shifts. the effect of these substances (a 23187) and of different temperatures on the ca2+ release from isolated vacuo ...19902159489
mutations that affect circadian rhythms in neurospora crassa can alter the reduction of cytochromes by blue light.we have examined membrane fractions from mutant strains of neurospora crassa that have altered responses to blue light or have altered circadian rhythms. using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. the poky strain was found to have high concentrations of photoreducible cytochrome c. after removal of this cytochrome, however, t ...19902151931
premeiotic instability of repeated sequences in neurospora crassa.maintenance of a steamlined genome is probably important to a free-living fungus. the period between fertilization and karyogamy in the life cycle of neurospora and related fungi provides an ideal time for "genome-cleaning". premeiotic intrachromosomal recombination deletes tandem repeats at high frequency in both homothallic and heterothallic filamentous ascomycetes. this eliminates excess copies of tandemly repeated genes and at the same time favors their homogenization. heterothallic fungi su ...19902150906
circadian rhythms in neurospora crassa: biochemistry and genetics. 19902147375
stearolic acid lengthens the condition period of neurospora crassa carrying the cel mutation. 19902145586
genetic coregulation of longevity and antioxienzymes in neurospora crassa.further analysis of a model of the biochemical genetics of cellular longevity in neurospora crassa confirms and amplifies the hypothesis that antioxienzymes and lifespans are genetically co-regulated. the model consists of seven classes of closely related strains with genetically determined median lifespans ranging from 7 to 90 days and differing by about 15-day intervals. the nuclear gene mutations age- and age+ respectively decrease and increase both lifespans and the constitutive enzyme activ ...19902143165
genes responsive to the alteration of polyamine biosynthesis in neurospora crassa.wild-type neurospora crassa grown in minimal medium was exposed to -difluormethyl ornithine (dfmo), a specific inhibitor of ornithine-decarboxylase (odc-ase) activity. protein-synthesis rates impaired by dfmo were restored by the addition of spermidine. the pattern on sds-acrylamide gels displayed three newly synthesized polypeptides, p27, p31 and p99 after dfmo action in the absence of exogenous polyamine. the odc-ase mutant (spe-1) grown in spermidine-supplemented medium did not show an induce ...19902139806
a kinetic study of the interaction between glycogen and neurospora crassa branching enzyme.the interaction of neurospora crassa branching enzyme (1,4-alpha-d-glucan:1,4-alpha-d-glucan 6-alpha-(1,4-alpha-glucano)-transferase) [ec] with substrate glycogen or amylopectin was studied by affinity electrophoresis. by this method, the dissociation constants (k) of the branching enzyme for oyster glycogen (cl-12.2, ocl-6.3) and for potato amylopectin (cl-20, ocl-12.8) were determined to be 13.3 mm and 0.355 mm, respectively. the affinity of the enzyme to the substrate glycogen increa ...19902139655
mode of action of glycogen branching enzyme from neurospora crassa.neurospora crassa branching enzyme [ec] acted on potato amylopectin or amylose to convert them to highly branched glycogen-type molecules which consisted of unit chains of six glucose units. the enzyme also acted on the amylopectin beta-limit dextrin, indicating that the enzyme acted on internal glucose chains as well as outer chains. by the combined action of n. crassa glycogen synthase [ec] and the branching enzyme, a glycogen-type molecule was formed from udp-glucose. in the ...19902139654
fast induction of translatable mrna by blue light in neurospora crassa wt: the wc-1 and wc-2 mutants are blind.after blue-light irradiation of neurospora crassa (wt) mycelia we observed an increase of about 13 translatable mrna species within a period of 30 min. the induction of translatable mrna species followed a specific temporal pattern which permitted the identification of four distinct classes. one of the translatable mrnas was induced in less than 2 min, while the others showed lag periods of 5, 10 or 20 min from the beginning of illumination. the white collar mutants, wc-1 and wc-2, which do not ...19902138217
preparation of neurospora crassa chromosomes from a cell wall-less strain. 19892532324
secretion of an mr 60000 protein by benomyl-treated cells of neurospora the presence of the microtubule inhibitor benomyl at micron concentrations, cells of neurospora crassa wild type strain st. lawrence 74a were found to secrete high amounts of an mr 60 000 protein into the culture medium (about 35 micrograms/ml after a 12 h treatment). the secretion also occurred after treatment with the other antitubulin drugs carbendazim (mbc), nocodazole, thiabendazole, and griseofulvin. this secretion is apparently induced by the specific action of benomyl on n. crassa bet ...19892534075
nuclear gene for mitochondrial leucyl-trna synthetase of neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.we have isolated and characterized the nuclear gene for the mitochondrial leucyl-trna synthetase (leurs) of neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. we have purified mitochondrial leurs protein, determined its n-terminal sequence, and used this sequence information to identify and isolate a full-length genomic dna clone. the 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced ...19892574823
isolation and characterization of a laccase-derepressed mutant of neurospora crassa.laccase from the ascomycete neurospora crassa is an inducible secretory enzyme. production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. the lah-1 mutation is mapped between nit-2 and leu-3 on linkage group i, and it behaved as a recessi ...19892553675
classical and molecular genetic analyses of his-3 mutants of neurospora crassa. ii. southern blot analyses and molecular mechanisms of mutagenicity.previous studies (overton et al., mutation res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3b mutants. in the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. the restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-or23-1a and were ...19892530448
isolation and characterization of the tyrosinase gene from neurospora crassa.a precursor form of neurospora crassa tyrosinase has been identified by western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mrna. the molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. in order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. poly(a) rna isolated from tyrosinase-induced cultures of n. crassa was used as a template for cdna synthes ...19892529259
evidence for an essential histidine residue in the neurospora crassa plasma membrane h+-atpase.the neurospora crassa plasma membrane h+-atpase is rapidly inactivated in the presence of diethyl pyrocarbonate (dep). the reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 m-1.min-1 at ph 6.9 and 25 degrees c. the difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of n-carbethoxyhistidine. no change in the absorbance of the inhibited atpase at 278 nm or in the number of ...19892528992
an ethidium bromide induced mutant of neurospora crassa defective in mitochondrial dna.slow growing mutants of neurospora crassa were obtained by ethidium bromide treatment of the wild type strain. a particular mutant er-3 showed stopper phenotype accompanied by deficient cytochrome spectra. the mutant showed an altered restriction pattern of the mtdna which indicated a deletion of 25,000 bp. the phenotype of the ethidium bromide induced mutant er-3 seem to be related to the loss of several essential genes due to a deletion in its mtdna.19892534062
molecular and classical genetic analyses of his-3 mutants of neurospora crassa. i. tests for allelic complementation and specific revertibility.a collection of 81 his-3 mutants of neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. in these studies, the linearity of the complementation map of the his-3 cistron (webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. in the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment w ...19892529437
a cycloheximide-inducible gene of neurospora crassa belongs to the cytochrome p-450 superfamily. 19892529480
the vacuolar atpase of neurospora crassa contains an f1-like structure.we have explored the structure and subunit composition of the vacuolar atpase of neurospora crassa by investigating the effects of nitrate. inhibition of enzyme activity by nitrate was correlated with dissociation of a complex of peripheral polypeptides from the integral membrane part of the enzyme. surprisingly, this nitrate-induced release of subunits occurred only when nucleotides such as adp, atp, or itp were present. atpase inhibitors that have been proposed to act at the active site preven ...19892527854
calcium activates an electrogenic proton pump in neurospora plasma membrane.calcium ionophoresis into coenocytic cells of neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. this is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi.198916666998
isolation of a gene that down-regulates nitrate assimilation and influences another regulatory gene in the same system.glutamine is the preferred source of nitrogen of neurospora crassa. in its presence and that of the gene product of ms5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. in the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. we report here the isolation, from a cosmid bank, of a gene that complements ms5 and can a ...19892528690
molecular cloning and regulatory analysis of the arylsulfatase structural gene of neurospora crassa.the ars-1+ gene of neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. the ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. cotransformation of an n. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by as ...19892528685
epistasis, photoreactivation and mutagen sensitivity of dna repair mutants upr-1 and mus-26 in neurospora crassa.double mutants were constructed combining mus-26, formerly designated uvs-(sa3b), with other uv-sensitive mutants. tests of sensitivity of these double mutants to uv and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. the uv dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 j/m2. the same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. photoreactivation of uv damage ...19892528064
mutations in nuclear gene cyt-4 of neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial rnas.the nuclear cyt-4 mutants of neurospora crassa have been shown previously to be defective in splicing the group i intron in the mitochondrial large rrna gene and in 3' end synthesis of the mitochondrial large rrna. here, northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial rna processing pathways, including those for cob, coi, coii and atpase 6 mrnas, as well as mitochondrial trnas. defects in these pathways include inhibition of 5' and 3' ...19892478417
ubiquitin expression in neurospora crassa: cloning and sequencing of a polyubiquitin gene.we have cloned and sequenced a polyubiquitin gene from neurospora crassa that is organized in a four repeat-tandem array. the first repeat contains a small intron and the last is fused to an extra glutamine codon. in northern blots, two rna species of 1.3 kb and 0.7 kb hybridize to the isolated clone. the larger ubiquitin (ubi) transcript accumulates after partial inhibition of protein synthesis with cycloheximide, and the smaller one preferentially accumulates in conidia after germination. unex ...19892549509
premeiotic change of nucleolus organizer size in neurospora.we have investigated the heritability of nucleolus organizer region (nor) size in neurospora crassa. by pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" nors and the size of a small interstitial nor. tetrad analysis revealed that changes in nor size occur frequently in the sexual phase. moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. unexpectedly, in ...19892527181
inositol trisphosphate induces calcium release from neurospora crassa vacuoles.inositol 1,4,5-trisphosphate is known to release calcium ions from intracellular stores thought to be parts of endoplasmic reticulum in animal cells. in neurospora crassa, however, inositol 1,4,5-trisphosphate acts on vacuoles stimulating a calcium efflux with a km of 5.28 microm. the calcium release is inhibited effectively by dantrolene. these results were obtained by applying two independent methods, measuring calcium binding to fura-2 and loading vacuoles with 45ca.19892527035
deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of neurospora crassa.deoxyribonucleoside triphosphate (dntp) levels were measured in wild type neurospora and nine mutagen-sensitive mutants, at nine different genes. eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dntps. two patterns were seen. five of the mutants had altered ratios of dntps, with relatively high levels of datp and dgtp and low levels of dctp, but changes in the dttp/dctp ratio did not correlat ...19892527032
studies on the active site of the neurospora crassa plasma membrane h+-atpase with periodate-oxidized nucleotides.the neurospora crassa plasma membrane h+-atpase is inactivated by the periodate-oxidized nucleotides, oatp, oadp, and oamp, with oamp the most effective. inhibition of the atpase is essentially irreversible, because sephadex g-50 column chromatography of the oamp-treated atpase does not result in a reversal of the inhibition. inhibition of the atpase by oamp is protected against by the h+-atpase substrate atp, the product adp, and the competitive inhibitors tnp (2',3'-o-(2,4,6-trinitrocyclohexad ...19892545685
isolation of nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in neurospora crassa.expression of nitrate reductase in neurospora crassa requires the positive action of nit-4, a pathway-specific regulatory gene, which mediates nitrate induction. we report the molecular cloning of the nit-4 gene and present results which suggest that the nit-4 gene is constitutively expressed to yield a low-abundance 2.2-kilobase transcript. these results indicate that the nit-2 major control gene and the nit-4 pathway-specific control gene independently regulate the expression of the nitrate as ...19892567729
effect of the uvs-2 allele of neurospora crassa on the mutagenic potency of two n-hydroxylaminopurines and 2-aminopurine in the ad-3 forward-mutation test.the mutagenic potencies of 3 purine analogs were determined in the ad-3 forward-mutation test in growing cultures of heterokaryon 59 (h-59), a nucleotide excision repair-deficient (uvs-2/uvs-2) 2-component heterokaryon of neurospora crassa. two n-hydroxylaminopurines, 2-amino-6-n-hydroxylaminopurine (aha) and 6-n-hydroxylaminopurine (hap), were potent and strong mutagens, respectively, whereas 2-aminopurine (ap) was a moderate mutagen. dose-response curves showed that aha and hap were about equa ...19892526296
the response time of transcription and translation of the leu-2 gene of neurospora to its inducer, alpha-isopropylmalate, approaches the permissible minimum.the rate of transcription and translation of the leu-2 gene of neurospora crassa was measured after induction by alpha-isopropylmalate. little message of enzyme was found before inducer addition but transcription in the lower eukaryote was found well underway within five minutes after inducer addition, followed in a minute or two by the appearance of functional enzyme. the timing was close to the limit set by rna synthesis and ribosome procession. as a consequence, it seems unlikely that travers ...19892525903
use of transformation to make targeted sequence alterations at the am (gdh) locus of neurospora.specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus of am (nadp-specific glutamate dehydrogenase) of neurospora crassa. two approaches have been successful. one approach used am+-containing vectors capable of integrating at any site in the genome. this technique was used to introduce a specific 700 bp insertion near the am locus and to replace chromosomal sequences near am with plasmid dna. efficiency was low, however, and ma ...19892549376
early response and induced tolerance to cycloheximide in neurospora crassa.incubation of neurospora crassa mycelia with low doses of cycloheximide induces the expression of several genes. after 6 h in the presence of cycloheximide, mycelia become tolerant to further additions of the drug and the rate of protein synthesis exhibits a lower sensitivity to it. the polypeptide pattern is indicative of a stress situation.19892528413
effects of heat shock on the induction of mutations by chemical mutagens in neurospora crassa.preheating of neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 x 10(7) conidia/ml 0.067 m kh2po4-na2hpo4 (ph 7.0) buffer to be treatment at 43 degrees c for 60 min. when protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state ...19892525670
uptake, intracellular binding, and excretion of polyamines during growth of neurospora neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. we wished to know what determines these pool sizes. in the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. in a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. however, this was true only after an intracellular "sink," with a c ...19892524999
independent transfer of mitochondrial plasmids in neurospora the ascomycete fungus neurospora, the distribution of homologous mitochondrial plasmid dnas in different species and among mitochondrial types of n. crassa suggests that these molecules have moved between lineages of clonally propagated mtdna. here we report direct evidence for independent inheritance of mitochondrial plasmids by sexual reproduction which may help explain the distribution of these molecules among mitochondrial lineages.19892524667
fusion of the mitochondrial outer membrane: use in forming large, two-dimensional crystals of the voltage-dependent, anion-selective channel protein.phospholipase a2 induces crystallization of the channel protein, vdac (also called mitochondrial porin), in the outer membrane of neurospora crassa mitochondria. the channel crystals formed in native membranes typically contain a few hundred unit cells. to increase the size of these membrane crystals for low-contrast electron microscopic imaging and diffraction studies, fusion of the isolated mitochondrial outer membranes was attempted before and after phospholipase treatment. successful fusion ...19892470408
luminescence emission from neurospora copper metallothionein. time-resolved studies.the luminescence lifetime of cu-metallothionein from the fungus neurospora crassa has been studied by the frequency-domain emission technique. lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. binding of hg(ii) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. no quenching by oxygen is found for the hg(ii)-cu-metallothionein adduct. by analogy to model compoun ...19892528343
dna sequence, organization and regulation of the qa gene cluster of neurospora neurospora, five structural and two regulatory genes mediate the initial events in quinate/shikimate metabolism as a carbon source. these genes are clustered in an 18 x 10(3) base-pair region as a contiguous array. the qa genes are induced by quinic acid and are coordinately controlled at the transcriptional level by the positive and negative regulators, qa-1f and qa-1s, respectively. the dna sequence of the entire qa gene cluster has been determined and transcripts for each gene have been ma ...19892525625
identification of an arginine carrier in the vacuolar membrane of neurospora crassa.a number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of neurospora crassa. the guanido side chain and l-configuration were found to be important for recognition by the arginine carrier. based upon the specificity of recognition, a reactive arginine derivative (n alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. the latter decomposes ...19892523392
identification and electron microscopic analysis of a chaperonin oligomer from neurospora crassa mitochondria.a 7-fold symmetric particle has been identified in neurospora crassa which is most probably the mitochondrial chaperonin. the particle, about 12 nm in diameter, appears in preparations of cytochrome reductase, and is shown to contain a 60 kd protein which cross-reacts with anti-groel antibodies. results of stem mass measurement suggest that the particle is composed of 14 subunits. a preliminary interpretation of the structure of the particle based on electron microscopy is given. its quaternary ...19892569968
premeiotic disruption of duplicated and triplicated copies of the neurospora crassa am (glutamate dehydrogenase) gene.premeiotic inactivation of duplicated sequences (the rip phenomenon of selker et al.) was studied by tetrad analysis using ectopic copies of am+ (coding for nadp-specific glutamate dehydrogenase) and a missense allele am3, coding for a distinctive form of the enzyme, at the normal locus. in duplication crosses either both gene copies were inactivated or neither. two inactivated am3 derivatives were shown to have undergone methylation and numerous base-pair changes, reflected in losses and gains ...19892529044
bioelectrorheological model of the cell. 2. analysis of creep and its experimental verification.the electrorheological model of the cell proposed in part 1 of this work was used to analyze changes in time of the shape of a cell acted on by a constant-amplitude external alternating electric field, with lossiness of the media taken into account. shear stress in the cell membrane was determined. this model was then subjected to preliminary experimental verification using neurospora crassa (slime) spheroplasts subjected to an external alternating electric field of constant frequency (3 mhz) an ...19892533955
proton nmr studies of a metallothionein from neurospora crassa: sequence-specific assignments by noe measurements in the rotating frame.sequential 1h nmr assignments of a metallothionein from neurospora crassa have been accomplished by the combined use of cosy, 2qf-cosy, hohaha, and rotating-frame noe experiments. all potentially observable resonances were assigned except for the epsilon-nh3 group of the c-terminal lysine. 1h noes, when observed in the laboratory frame and at 500-mhz spectrometer frequency, were negligible in this protein due to the inherent rotational correlation time of the molecule. this difficulty was circum ...19892525920
two kinds of "recombination nodules" in neurospora crassa.two morphological types of recombination nodules, termed early and late, are recognized in neurospora crassa. eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transi ...19892526043
a morphological and genetic analysis of conidiophore development in neurospora crassa.the filamentous fungus neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. these conidia are derived from differentiated aerial structures called conidiophores. the process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (sem) and specific fluorescent probes. the first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeat ...19892524423
change in chromosome number associated with a double deletion in the neurospora crassa mitochondrial chromosome.the mitochondrial genome of neurospora is usually found in a single covalently closed circular 62-kbp dna molecule. we report here that the mitochondrial genome of a phenotypic revertant of a stopper mutant (stp-ruv) is contained primarily in two separate, nonoverlapping, autonomously replicating circular chromosomes. the circles, one about 21 kbp and the other somewhat less than 36 kbp are derived from the most frequent classes of recombinant chromosomes (21 and 41 kbp) in the chromosomal popul ...19892524420
characterization of nitrate reductase deficient mutants of chlorella sorokiniana.after x-ray irradiation, 13 mutants of chlorella sorokiniana incapable of using no(3) (-) as n source were isolated using a pinpoint method. using immunoprecipitation and western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. the latter strains contained different patterns of nitrate reductase partial reactions. all isolates were of the nia-type as indicated by the inducibility of purine hydroxylase i and by complementation of nitrate ...198916666622
isolation of a transposable element from neurospora crassa.a neurospora crassa strain from adiopodoumé, ivory coast, contains multiple copies of a transposable element, tad. the element was detected as a 7-kilobase insertion in two independently isolated spontaneous forward mutants of the am (glutamate dehydrogenase) gene. laboratory strains do not contain tad. all progeny from crosses of the adiopodoumé strain to laboratory strains contain multiple copies. when the element was inserted in am, target sequences of 14 and 17 base pairs were duplicated in ...19892538822
isolation and characterization of cadmium-resistant mutants of neurospora crassa.this study identified and characterized four cadmium-resistant mutants of neurospora crassa. one of these mutants maps to linkage group ii and the other three map to linkage group vii, whereas a naturally occurring resistant trait in a strain from japan resides at a distinct but unmapped locus. transport of cadmium into neurospora cells occurs by more than a single uptake system and involves both energy-dependent and -independent components. the resistant mutants transport cadmium in the same ma ...19892525066
immunological identification of the alternative oxidase of neurospora crassa mitochondria.neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. we used a monoclonal antibody to the alternative oxidase of the higher plant sauromatum guttatum to identify a similar set of related polypeptides (mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of ...19892524649
a small isoform of nadh:ubiquinone oxidoreductase (complex i) without mitochondrially encoded subunits is made in chloramphenicol-treated neurospora mitochondria of neurospora crassa grown in the presence of chloramphenicol a small form of nadh:ubiquinone reductase is made in place of the normal electron-transfer-complex i. this smaller enzyme has a molecular mass of approximately 350 kda and consists of (at least) 13 different subunits which are all synthesized in the cytoplasm. the complex i which is normally found in neurospora has a molecular mass of approximately 700 kda and consists of around 30 different subunits, of which at least ...19892523306
13n isotope studies of glutamine assimilation pathways in neurospora crassa.l-[amide-13n]glutamine in neurospora crassa is metabolized to [13n]glutamate by glutamate synthase and to [13n]ammonium by the glutamine transaminase-omega-amidase pathway. the [13n]ammonium released is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. most of the nitrogen is retained during cycling between glutamate and glutamine.19892522094
in vivo control of gluconeogenesis in wild-type neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.the rate of cycloheximide-resistant incorporation of carbon from [14c]alanine and [14c]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. the wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. cycloheximide-resistant 14c-incorporating activity was sensitive to carbon catabol ...19892522093
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. ii. more extensive genetic tests reveal an unexpectedly high frequency of multiple-locus mutations.more extensive genetic tests have been performed on a series of 832 x-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon (h-12) of neurospora crassa, reported earlier (webber and de serres 1965). using new tester strains and techniques for performing large-scale genetic tests (heterokaryon, dikaryon and trikaryon) to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of x-ray-induced ad-3 mutants. these ne ...19892521371
neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.a method is proposed for the purification of the neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on sds-polyacrylamide gel electrophoresis as e3, e1 and e2 with respective mr values of 54,000, 53,000 and 49,000. catalytic properties of the purified co ...19892521564
sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of neurospora crassa.marked differences in the pattern of sedimentation of cellular structures were observed after isopycnic centrifugation of crude cell-free preparations from the neurospora crassa wall-less 'slime' variant and mycelial wild-type strain. kinetic studies of particle sedimentation showed that the various types of subcellular components, as revealed by turbidity, uv absorption, polypeptide patterns, and chitin synthetase activity determinations, sediment independently of one another. an important feat ...19892521563
nad-specific glutamate dehydrogenase of neurospora crassa. cdna cloning and gene expression during derepression.the catabolic nad-specific glutamate dehydrogenase of neurospora crassa is one of the many enzymes regulated by carbon catabolite repression. to achieve an understanding of its regulation, cdna and genomic clones were isolated. total poly(a+) rna from derepressed cells was used for the construction of a cdna library in the expression vector, lambda gt11. by screening this library with a polyclonal antiserum against nad-specific glutamate dehydrogenase, a positive clone with a 0.9-kilobase insert ...19892521336
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