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interactions of neurospora crassa plasma membrane h+-atpase with n-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.the carboxyl group activating reagent n-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (eedq) interacts with the neurospora plasma membrane h+-atpase in at least three different ways. this reagent irreversibly inhibits atp hydrolysis with kinetics that are pseudo-first-order at several concentrations of eedq, and an appropriate transform of these data suggests that 1 mol of eedq inactivates 1 mol of the h+-atpase. inhibition probably involves activation of an atpase carboxyl group followed by a ...19862874829
the properties of arginine transport in vacuolar membrane vesicles of neurospora crassa.we have measured the uptake of arginine into vacuolar membrane vesicles from neurospora crassa. arginine transport was found to be dependent on atp hydrolysis, mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. the mg2+ concentration required for optimal arginine transport varied with the atp concentration so that maximal transport occurred when the mgatp2- concentration was at a maximum and the concentrations of free atp and mg2+ were a ...19862941421
efficient cloning of genes of neurospora crassa.we have constructed a genomic library of neurospora crassa dna in a cosmid vector that contains the dominant selectable marker for benomyl resistance. the library is arranged to permit the rapid cloning of neurospora genes by either sib-selection or colony-hybridization protocols. detailed procedures for the uses of the library are described. by use of these procedures, a modest number of unrelated genes have been isolated. the cloning of trp-3, the structural gene for the multifunctional enzyme ...198616593723
genetical and molecular analyses of qa-2 transformants in neurospora crassa.neurospora crassa qa-2+ transformants from five different donor dna clones were analyzed by genetical and molecular techniques. none of the 32 transformants have the qa-2+ dna replacing the qa-2- gene in linkage group vii. in one transformant, the qa-2+ dna was inserted adjacent to the qa-2- gene. thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in lg vii. plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. ...19863015719
[nad+-kinase from neurospora crassa: isolation and properties].the nad+ kinase (ec 2.7.1.23) of the filamentous fungus n. crassa is localized in cytosol. the activity in the dialyzed cell free extract has a ph optimum 8.3; it utilizes only atp but not inorganic polyphosphates as a phosphoryl donor. a method for 200-fold purification of nad+ kinase with a 20% yield has been developed. the procedure includes 105000 g centrifugation, fractionation with (nh4)2so4, isoelectrofocusing in a ultrodex layer and preparative electrophoresis in polyacrylamide gel. the ...19863015266
cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of neurospora crassa and its use as a dominant selectable marker.we cloned the beta-tubulin gene of neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. the gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. the coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. intron position comparisons between the n. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. the mutatio ...19862946938
biochemical comparison of the neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. detailed kinetic comparison of the leucyl-trna synthetases.the cytoplasmic leucyl-trna synthetases were purified from a wild-type neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. a detailed steady-state kinetic study of the aminoacylation of the trnaleu from n. crassa by the purified synthetases was carried out. these enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. the kappm value for leucine was lowered by high atp concentrations and correspondingly the ...19862942399
heat shock protects germinating conidiospores of neurospora crassa against freezing injury.germinating conidiospores of neurospora crassa that were exposed to 45 degrees c, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees c. whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies t ...19862941411
regulation of amino acid synthetic enzymes in neurospora crassa in the presence of high concentrations of amino acids.ornithine carbamoyl transferase and leucine aminotransferase of neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. in the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mm range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. a cpc-1 mutant st ...19862943971
identification of dna repair and damage induced proteins from neurospora crassa.the response of neurospora crassa to dna damage induced by uv irradiation has been studied using two-dimensional polyacrylamide gel electrophoresis (2-d page). whole cell extracts of irradiated and untreated cultures were compared. five polypeptides that show changes in response to dna damage have been identified. several mutagen sensitive strains of neurospora were also tested for polypeptide changes on 2-d page. profiles of whole cell extracts of these mutant strains were compared to wild type ...19862943970
rearrangement mutations on the 5' side of the qa-2 gene of neurospora implicate two regions of qa-1f activator-protein interaction.transcriptional activation of the neurospora crassa qa genes normally requires the positive regulatory gene, qa-1f+, whose function is controlled by the inducer quinic acid and by the product of the negative regulatory gene, qa-1s+. the properties of qa-1f+ activator have been examined in transcriptional mutations of the qa-2 structural gene, in which activator-independent transcription of qa-2 (qa-2ai mutants) occurs in strains having a qa-1f- gene. seven qa-2ai mutants with dna rearrangements ...19862940595
pantothenate is required in neurospora crassa for assembly of subunit peptides of cytochrome c oxidase and atpase/atp synthase.one polypeptide subunit of cytochrome c oxidase (ec 1.9.3.1) and two subunits of the atpase/atp synthase (ec 3.6.1.34) in mitochondria of neurospora crassa are covalently modified with a derivative of pantothenic acid. in asexual spores of a pantothenate auxotroph of neurospora, deprivation of pantothenic acid blocked the increase of the specific activities of cytochrome c oxidase and the atpase above the basal activities in the dormant spores. under cellular panthothenate deprivation, all the s ...19862872672
oxidation of neurospora crassa glutamine synthetase.the glutamine synthetase of neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus fecl3 and by h2o2 plus feso4. the inactivation reaction was oxygen dependent, inhibited by mncl2 and edta, and stimulated in cell extracts by sodium azide. this inactivation could also be brought about by adding nadph to the cell extract. the alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acid ...19862872202
n-acetyl-l-glutamate synthase of neurospora crassa. characteristics, localization, regulation, and genetic control.n-acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. we have detected this enzyme in crude extracts of neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. t ...19862939069
are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in neurospora crassa?a triple albino mutant of neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10(-4)%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. this is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. however, it is impossible to exclude traces of carotenoid ...198624233735
elevation of alu i-induced frequencies of chromosomal aberrations in chinese hamster ovary cells by neurospora crassa endonuclease and by ammonium sulfate.the frequencies of chromosomal aberrations induced by the restriction endonuclease alu i (recognition site ag/ct) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from neurospora crassa (ec 3.1.30.1), or with ammonium sulfate in which the neurospora endonuclease is suspended. these data indicate that alu i does not produce dna single-strand breaks in the chromatin of living cells, which can be recognized by the neurospora endonuclease. the s ...19863010098
effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in neurospora crassa.the specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (ec 2.1.3.3) in neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. the increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. however, de ...19862939061
sequencing studies of icr-170 mutagenic specificity in the am (nadp-specific glutamate dehydrogenase) gene of neurospora crassa.the acridine half-mustard icr-170-induced reversion of the mutant am15, which has a single base-pair deletion, at a frequency of between 9 and 28 x 10(-6). in each of three classes of revertants, the mutagen had induced the insertion of a -g- -c- base pair at a -g-g- -c-c- site. the mutant am6, which has a single base pair insertion, is known to be revertible, with uv light, by deletion of a -g- -c- base pair at a -g-g-g- -c-c-c- site. this mutant reverted with icr-170 at a frequency of 0.1 x 10 ...19862423414
a missense mutation in the oxi-3 gene of the [mi-3] extranuclear mutant of neurospora crassa.we have determined the dna sequence of the oxi-3 gene and its 5' flanking region in the extranuclear [mi-3] mutant of neurospora crassa. the oxi-3 gene encodes subunit 1 of cytochrome c oxidase, a protein known to be altered in the [mi-3] mutant (bertrand, h., and werner, s. (1979) eur. j. biochem. 98, 9-18). when the sequence from [mi-3] was compared to previously published sequences of the same region of mtdna from wild-type n. crassa, a total of five differences was found. four of these diffe ...19863007516
the e35 stopper mutant of neurospora crassa: precise localization of deletion endpoints in mitochondrial dna and evidence that the deleted dna codes for a subunit of nadh dehydrogenase.two types of defective mitochondrial dna molecules with large deletions (5 kbp and 40 kbp) have previously been identified in the stopper mutant, e35, of neurospora crassa. the junction fragments spanning the deletion endpoints have now been cloned and sequenced, and their sequences compared with those of the corresponding wild-type fragments. we show that both types of defective mitochondrial dnas result from deletions of sequences flanked by short direct repeats, which are themselves parts of ...19863011426
a study of derepression of nad-specific glutamate dehydrogenase of neurospora crassa.transfer of neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic nad-specific glutamate dehydrogenase (nad-gdh), within 30 min of the shift. immunoprecipitation of in vivo pulse-labelled nad-gdh demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. derepression was shown to be under transcriptional control by using the rna synthesis ...19862944990
primary structure and spectroscopic studies of neurospora copper metallothionein.when neurospora crassa is grown in the presence of cu(ii) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. the molecule binds 6 g-atom of copper per mole protein (mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of neurospora metallothionein indicate the copper to be bound to cysteinyl residu ...19863011391
isolation and partial characterization of two antifungal proteins from barley.we have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, trichoderma reesei. using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. the first protein has a molecular weight of 30 000 and is identical to the 30 kda ...19863942788
structure of the channels in the outer mitochondrial membrane: electron microscopic studies of the periodic arrays induced by phospholipase a(2) treatment of the neurospora membrane.the channel proteins in outer membranes of neurospora crassa mitochondria spontaneously organize into periodic arrays when the membranes are dialyzed in the presence of soluble phospholipase a(2). electron microscopic images have been recorded at different electron doses from channel arrays in a variety of negative stains, as well as in vitreous ice. fourier or correlation averages are formed from image fields containing a few hundred unit cells. these averages can be subsequently classified by ...198619431637
structural studies of the vacuolar membrane atpase from neurospora crassa and comparison with the tonoplast membrane atpase from zea mays.the h+-translocating atpase located on vacuolar membranes of neurospora crassa was partially purified by solubilization in two detergents, triton x-100 and n-hexadecyl-n,n-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. two polypeptides of mr approximately equal to 70,000 and approximately equal to 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. radioactively labeled inhibitors of atpase activit ...19863079903
neurospora crassa ribosomal dna: sequence of internal transcribed spacer and comparison with n. intermedia and n. sitophila.using [32p]dna probes from a clone containing 17s, 5.8s and 26s rrna of neurospora crassa, the remainder of the repeat unit (ru) for ribosomal dna (rdna) has been cloned. combining restriction analysis of the cloned dna and restriction digests of genomic dna, the ru was found to be 8.7 kb. the nucleotide sequence was determined for the internal transcribed spacer (its) regions one and two, for 5.8s rrna and for portions of 17s and 26s rrnas immediately flanking the its regions, and compared to t ...19863021585
loss of temperature compensation of circadian period length in the frq-9 mutant of neurospora crassa.a new circadian clock mutant of neurospora crassa has been isolated, whose most distinctive characteristic is the complete loss of temperature compensation of its period length. the q10 of the period length was found to be equal to about 2 in the temperature range from 18 degrees to 30 degrees c. the period length was also found to be dependent on the composition of the medium, including the nature and concentration of both the carbon source and the nitrogen source. although the rate of the cloc ...19862980965
phase shifting of the circadian conidiation rhythm in neurospora crassa by calmodulin antagonists.the effects of chemicals capable of antagonizing the functions of calmodulin, such as trifluoperazine, chlorpromazine, imipramine, alprenolol, w7, and w13, on the circadian conidiation rhythm of neurospora crassa were examined. trifluoperazine, at a 30-microm concentration, was most effective in shifting the phase of the conidiation rhythm and caused a maximum phase delay at circadian time (ct) 6 and maximum phase advance at ct 9. chlorpromazine was less effective than trifluoperazine, and a 300 ...19862980964
amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of neurospora crassa.neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. the nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. the nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. many new nit ...19862965995
the mitochondrial dna of neurospora crassa: deletion by intramolecular recombination and the expression of mitochondrial genes. 19862952110
genetic characterization of the mutagenic activity of environmental chemicals at specific loci in two-component heterokaryons of neurospora crassa. 19862944123
magnification of rrna gene number in a neurospora crassa strain with a partial deletion of the nucleolus organizer.some progeny from crosses between the neurospora crassa translocation strain t(il----vl)oy321 and normal sequence n. crassa strains are duplication strains with a partial deletion of the nucleolus organizer. despite the deletion, these progeny are viable and produce a functional nucleolus. quantification of rrna gene number in these deletion progeny demonstrated a significant loss of rrna genes, down to 60% of the parental wild-type level. initially, all of these reduced nucleolus organizer (rno ...19862938894
inhibitory effect of the partially resolved coordination isomers of chromic desferricoprogen on coprogen uptake in neurospora crassa.two partially resolved chromatographic fractions of geometrical and optical isomers of the chromic complexes of desferricoprogen, a siderophore from neurospora crassa, were obtained from high-pressure liquid chromatography on a reverse-phase matrix. the first fraction was identified as a cis complex with a 20% diastereomeric excess of the lambda isomer. the second fraction was identified as a mixture of several of the possible trans isomers with a net 20% diastereomeric excess of the delta isome ...19862934378
characterization of inl+ transformants of neurospora crassa obtained with a recombinant cosmid-pool.we constructed a neurospora crassa gene library in a cosmid vector and used the cosmid-pool dna to transform an inl, rg neurospora crassa strain to inositol prototrophy. the inl+ colonies obtained in this experiment proved to be integrative type transformants. genetic analysis revealed that the integration event occurred at or near the inl locus. in one of the transformants the inl+ trait exhibited mitotic and meiotic instability. in hybridization experiments free plasmids were detected in the f ...19862834083
conservation of a long open reading frame in two neurospora mitochondrial plasmids.the nucleotide sequence of a specific region of the mitochondrial plasmid from the neurospora intermedia varkud-lc strain was determined. analysis of the sequence revealed the presence of a long (up to 710 amino acids) orf. this orf is almost identical to a previously characterized orf in the mitochondrial plasmid from the neurospora crassa mauriceville-lc strain. when the orfs from the two plasmids are compared over their entire length of 2,133 bp, only 34 nucleotide substitutions are found (gr ...19862832686
light-regulated protein and poly(a)+ mrna synthesis in neurospora crassa.we have examined the effect of illumination upon the patterns of protein synthesis in the filamentous ascomycete neurospora crassa by pulse labelling and two-dimensional gel electrophoresis. light did not affect overall rates of protein synthesis but did induce the synthesis of six novel polypeptides whose appearance followed a temporally regulated pattern. when translation products of mrna from illuminated cultures and dark control cultures were compared it was found that the synthesis of five ...19852868891
structural studies on neurospora rna polymerases and associated proteins.extensive structural homology between the three nuclear rna polymerases of neurospora crassa has been observed by peptide and immunological analyses. within each polymerase, we found structural similarity between subunits in the 65- to 75-kda range and one of the two large subunits. we observed also that polymerase ii, as isolated, is associated with a 700-kda complex of 12 polypeptides which is localized in the nucleus. a 75-kda subunit of the 700-kda complex was found to be structurally relate ...19852933407
purification of the three nuclear rna polymerases from neurospora crassa.nuclear rna polymerases i, ii, and iii from neurospora crassa have been purified 3,000-, 1,500-, and 10,000-fold, respectively, by a procedure that minimizes proteolysis of the 220-kda subunit of polymerase ii. the neurospora enzymes resemble, in polypeptide composition, the corresponding polymerases isolated from other eukaryotes. the 220-kda subunit of neurospora polymerase ii cross-reacts with antisera directed against the 220-kda subunits of type ii polymerases from drosophila and wheat germ ...19852933406
autogenous regulation of the positive regulatory qa-1f gene in neurospora crassa.in neurospora crassa, the qa-1f regulatory gene positively controls transcription of all genes in the quinic acid (qa) gene cluster. qa-1f is transcribed at a low, uninduced level but is subject to strong (50-fold), autogenous regulation as well as to control by the negative regulatory gene, qa-1s, and the inducer quinic acid. cloned qa-1f dna sequences hybridize to two related mrnas of 2.9 and 3.0 kilobases. when wild-type (qa-1f+) cultures are transferred to inducing conditions, qa-1f mrna inc ...19852943985
evidence for a pterin-derivative associated with the molybdenum cofactor of neurospora crassa nitrate reductase. 19852937070
recessive mutations from natural populations of neurospora crassa that are expressed in the sexual diplophase.wild-collected isolates of neurospora crassa shear and dodge were systematically examined for recessive mutations affecting the sexual phase of the life cycle, which is essentially diploid. seventy-four of 99 wild-collected isolates from 26 populations in the united states, india and pakistan carried one or more recessive mutations that reduced fertility significantly when homozygous; mutations affecting spore morphology were also detected. limited complementation tests indicate that most of the ...19852933298
dna methylation at asymmetric sites is associated with numerous transition mutations.we describe two unusual 5s rna regions from neurospora crassa that are tightly linked. sequence analysis suggests that these genes or pseudogenes, which we designate zeta (zeta) and eta (eta), arose by a 794-base-pair tandem duplication followed by hundreds of exclusively cytosine to thymine mutations. the duplication was most likely generated by nonhomologous recombination involving a dna segment having a striking purine-pyrimidine strand asymmetry. restriction analysis of genomic dna from tiss ...19852415981
depression of enzyme synthesis in response to arginine limitation in neurospora crassa.ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in neurospora and show derepression in response to limitation of any one of a number of amino acids. the effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. depression occurred at arginine concentrations sufficient to allow normal rates ...19852936866
a new ultraviolet-light sensitive mutant of neurospora crassa with unusual photoreactivation property.a mutant, uvs-(sa3b), which shows high sensitivity to uv light segregated among the progeny in a back-cross of a presumptive mms-sensitive mutant to a wild-type strain. at 37% survival, this mutant was approximately 5 times more sensitive to uv and also 6 times more sensitive to 4-nqo than the wild type. but it was only slightly sensitive to gamma-ray, mms, mnng, mtc and histidine. it showed an unusual photoreactivation response. its time course of photorecovery was similar to the photoreactivat ...20132933585
isolation and structural organization of the neurospora crassa copper metallothionein gene.the neurospora crassa copper metallothionein gene was cloned and its complete nucleotide sequence is reported. enriched metallothionein mrna was used as a template for cdna synthesis, primed by a metallothionein-specific, synthetic undecanucleotide. the sequence of the cdna obtained allowed the synthesis of a unique 21-mer which was used to screen a genomic dna library of n. crassa. in agreement with the published amino acid sequence, the gene codes for a polypeptide 26 amino acid residues in le ...19852932331
general method for cloning neurospora crassa nuclear genes by complementation of mutants.we have developed a sib selection procedure for cloning neurospora crassa nuclear genes by complementation of mutants. this procedure takes advantage of a modified n. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of dna with recombinant plasmids containing the n. crassa qa-2+ gene. here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. the identities of the putative c ...19852942762
neurospora crassa and s1 nuclease cleavage in hsp 83 gene chromatin.we have examined the distribution of neurospora crassa and s1 nuclease cleavage products in the chromatin of the hsp 83 heat shock gene from the drosophila melanogaster cytogenetic locus 63 bc. both of these nucleases generate double strand breaks in chromatin at specific sites close to the 5' end of the hsp 83 gene. with n. crassa nuclease we observe one major 5' fragment which is derived from nuclease cleavage in a dna segment mapping approximately 120 base-pairs from the beginning of the tran ...19852995684
a regulatory protein for orthophosphate-regulated cyclic phosphodiesterase in neurospora crassa.a regulatory protein for orthophosphate-regulated cyclic phosphodiesterase (cpdase) was detected in mycelial extracts of neurospora crassa. the protein, designated neucrassin, was precipitated by ammonium sulfate between 60 to 100% saturation, and fractionated by gel filtration through a tsk-gel column. the molecular weight was estimated to be 65,000. neucrassin inhibited the hydrolyzing activity of cpdase for cyclic 3',5'-amp in the presence of mncl2 in a noncompetitive manner, whereas it stimu ...19852992492
rna splicing in neurospora mitochondria. defective splicing of mitochondrial mrna precursors in the nuclear mutant cyt18-1.cyt18-1 (299-9) is a nuclear mutant of neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rrna intron. in the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mrna introns. two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit i (coi) gene, which contains four introns. we found that splicing of both cob ...19852413216
identification of the polypeptide encoded by the urf-1 gene of neurospora crassa mtdna.two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. the peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (urf)1 of the neurospora crassa mitochondrial genome. specific antisera to the synthetic peptides were produced. the antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. the mitochondrial origin of this polypeptide was verified by in vivo ...19853160590
accurate transcription of cloned neurospora rna polymerase ii-dependent genes in vitro by homologous soluble extracts.we have developed soluble extracts from neurospora crassa capable of accurately initiating the transcription of cloned neurospora protein-encoding genes by rna polymerase ii in vitro. the genes encoding glutamate dehydrogenase (am) and histones h3 and h4 were transcribed by the extracts, and transcription was sensitive to alpha-amanitin at 1 mg/ml. the 5' heterogeneity of the in vitro initiation reactions was highly specific. of the 17 transcription initiation sites within the inducible qa gene ...19852991927
mitochondrial translation of subunits of the rotenone-sensitive nadh:ubiquinone reductase in neurospora crassa.the rotenone sensitive nadh:ubiquinone was isolated from mitochondria of neurospora crassa as a monodisperse preparation with the apparent mol. wt. in triton solution of 0.9 x 10(6). the enzyme is composed of at least 22 subunits with apparent mol. wts. in sds between 70 and 11 kd. six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35s]methionine in the presence of cycloheximide. these subunits ...19852933252
a water-soluble form of porin from the mitochondrial outer membrane of neurospora crassa. properties and relationship to the biosynthetic precursor form.mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water-soluble form. this water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. the proportion of dimers increased with decreasing porin concentration during conversion. water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. whereas the latter form had no specific requirements for t ...19852989279
blue light-reducible cytochromes in membrane fractions from neurospora crassa.we have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from neurospora crassa. light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. in plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. addition of riboflavin ...198516664261
gene disruption by transformation in neurospora crassa.to establish conditions which might permit deliberate gene disruptions in neurospora crassa, we studied transformation with linear dna fragments. the transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid dna. however, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming dna took place in nonhomologous regions. it was also found that multiple int ...19853160929
structural gene for ornithine decarboxylase in neurospora crassa.to define the structural gene for ornithine decarboxylase (odc) in neurospora crassa, we sought mutants with kinetically altered enzyme. four mutants, pe4, pe7, pe69, and pe85, were isolated. they were able to grow slowly at 25 degrees c on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees c. the mutants did not complement with one another or with odc-less spe-1 mutants isolated in earlier studies. in all of the mutants isolated to date, the mutations ...19853162095
effect of phosphate levels on the synthesis of acid phosphatases (ec 3.1.3.2) in neurospora crassa. 19853161781
distinct roles of putrescine and spermidine in the regulation of ornithine decarboxylase in neurospora crassa.we wished to identify metabolic signals governing changes in ornithine decarboxylase (l-ornithine carboxy-lyase, ec 4.1.1.17) activity in neurospora crassa. by manipulations of the ornithine supply and by the use of inhibitors of the polyamine pathway, we found that spermidine negatively governs formation of active ornithine decarboxylase and that putrescine promotes inactivation of the enzyme. direct addition of putrescine or spermidine to cycloheximide-treated cells confirmed the role of putre ...19853159019
heat shock response of neurospora crassa: protein synthesis and induced thermotolerance.at elevated temperatures, germinating conidiospores of neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 mr and one minor heat shock protein of 30,000 mr. postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 mr and a minor heat shock protein of 34,000 mr. the three heat shock proteins of lower molecular weight are associated with mitochondria. this exclusive ...19853158641
evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of neurospora crassa and compared localization with the pyruvate dehydrogenase complex.a 2-oxoglutarate dehydrogenase complex activity is demonstrated in neurospora crassa mitochondria. a submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. in contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.19852932166
preparation of functional group analogs of unsaturated fatty acids and their effects on the circadian rhythm of a fatty-acid-deficient mutant of neurospora crassa.functional group analogs of oleic, linoleic and linolenic acids were prepared by coverting their double bonds to dibromo, cyclopropyl, epoxy, methoxy, and, in the case of oleic acid, hydroxy groups. these compounds were supplemented to the bd csp cel strain of the mold neurospora crassa. the cel mutation confers a partial requirement for saturated fatty acids and, also, perturbs the circadian rhythm of spore formation. for example, the period of bd csp cel's rhythm is dramatically lengthened upo ...20082931204
acetylglutamate kinase-acetylglutamyl-phosphate reductase complex of neurospora crassa. evidence for two polypeptides.mutations at the arg-6 locus in neurospora crassa are divided into two complementation groups (a and b) and a third noncomplementing group. there are many suppressible nonsense mutations among mutants in complementation group b and one in the noncomplementing group; no nonsense mutations exist among mutants in complementation group a (davis, r. h., and weiss, r. l. (1983) mol. gen. genet. 192, 46-50). we show here that the mutants are defective in either or both of two enzymes of arginine biosyn ...19852987210
evidence for a common siderophore transport system but different siderophore receptors in neurospora crassa.uptake and competition experiments were performed with neurospora crassa and penicillium parvum by using 14c-labeled coprogen and 55fe-labeled ferrichrome-type siderophores. several siderophores of the ferrichrome family, such as ferrichrome, ferricrocin, ferrichrysin, and tetraglycyl-ferrichrome as well as the semisynthetic ferricrocin derivatives o-(phenyl-carbamoyl)-ferricrocin and o-(sulfanilyl-carbamoyl)-ferricrocin were taken up by n. crassa. the ferrichrome-type siderophores used vary in ...19852985545
isolation and characterization of genes differentially expressed during conidiation of neurospora crassa.a neurospora crassa genomic dna library was screened with a cdna probe enriched in sequences expressed in conidiating cultures. clones were isolated that preferentially hybridized to this probe versus a second cdna probe complementary to polyadenylated rna isolated from mycelia. twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. eight transcripts were present in higher levels in conidiating cultures than in ...19853157864
identification and chromosomal distribution of 5s rrna genes in neurospora crassa.the 5s rrna genes of neurospora crassa, unlike those of most organisms, are not tandemly arranged, and they are found imbedded in a variety of unique sequences. the 5s rrna regions of most of the genes are of one type, alpha; however, several other "isotypes" (beta, gamma, delta, zeta, and eta) are also found. we asked whether neurospora 5s rrna genes are dispersed on a chromosomal scale and whether genes of different isotypes are spatially segregated. we identified, by dna sequencing, 5s rrna g ...19853157192
the 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of neurospora crassa is zn2+-dependent.we have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from neurospora crassa. progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-d-arabino-heptulosonate 7-phosphate, but not by the cofactor nad+. full activity was restored at zn2+ concentrations as low as 0.05 nm. atomic absorption data indicated that the intact enzyme co ...19853157372
unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient neurospora crassa mutant.the fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. to test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the p ...19853156558
gamma-ray induced ageing mutants of neurospora crassa: response to some antioxidants and chloramphenicol. 19852932387
omega-amidase pathway in the degradation of glutamine in neurospora crassa.evidence for the participation of the glutamine transaminase-omega-amidase pathway in the utilization of glutamine in neurospora crassa was obtained. its participation is indicated by the in vitro activities of glutamine transaminase and omega-amidase, the in vivo accumulation of alpha-ketoglutaramate when an inhibitor of transamidases is present, and the inhibition by aminooxyacetic acid and 6-diazo-5-oxo-l-norleucine of the ammonium excreted in the presence of glutamine by a mutant strain that ...19852857167
the neurospora mitochondrial genome: the region coding for the polycistronic cytochrome oxidase subunit i transcript is preceded by a transfer rna gene.we have sequenced a 682 bp fragment of neurospora crassa mitochondrial dna to complete the sequence between the gene for cytochrome b and the unassigned reading frame, urf u. the sequence contains a gene for a cysteine trna. the 5' end of the 6 kb polycistronic transcript of cytochrome c oxidase subunit 1 is immediately downstream from this trna. this shows that also in fungal mitochondria trnas can be used as processing signals, whereas palindromic sequences containing double pst i sites, also ...19852981709
a pantothenate derivative is covalently bound to mitochondrial proteins in neurospora crassa.the presence of protein-bound pantothenate in neurospora crassa was investigated by labelling a pantothenate auxotroph (pan-2) with [14c]pantothenate and examining mycelial homogenates on dodecyl sulfate/polyacrylamide gels. five peaks of radioactivity were found, with apparent molecular masses of 200, 140, 22, 19, and 9 kda. the 200-kda peak was identified as fatty acid synthetase, based on its absence in a fatty acid synthetase mutant. the 22-kda and 19-kda peaks co-purified with mitochondrial ...19853155682
carotenoid synthesis in neurospora crassa. 19853160913
rna processing in neurospora crassa mitochondria: transfer rnas punctuate a large precursor transcript.the pattern of transcripts arising from a large contiguous portion of the neurospora crassa mitochondrial genome and the processing of these transcripts have been investigated. evidence is presented for the transcription of a single, at least 12.5 kb, precursor rna that contains sequences corresponding to the apocytochrome b (cob), trnacys, cytochrome oxidase subunit 1 (co i), trnaarg and unidentified reading frame (urf) 1 genes. the two trna sequences serve as punctuation signals for the cleava ...19853160579
mutagenicity of 2-aminopurine, 6-n-hydroxylaminopurine, and 2-amino-n6-hydroxyadenine in neurospora crassa.these data from our experiments with 3 purine analogs reveal striking differences in mutagenic potency. it seems highly likely that these analogs substitute readily for adenine and that they cause mutations in the main part, and in the case of aha perhaps predominantly, by mispairing with cytosine. the most potent mutagens are those with the hydroxylamino group at the c6 position (aha and hap). of these, the most potent is the analog with an amino group in the c2 position (aha). the most interes ...19853158304
inducible beta-oxidation pathway in neurospora crassa.an inducible beta-oxidation system was demonstrated in a particulate fraction from neurospora crassa. the activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. the induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. since an acyl-coenzyme a (coa) dehydrogenase was detected instead of acyl-coa oxidase, the former enzyme seems to catalyze the first step of the beta-oxi ...19853155714
circadian rhythms in neurospora crassa: interactions between clock mutations.mutations at four loci in neurospora crassa that alter the period of the circadian rhythm have been used to construct a series of double mutant strains in order to detect interactions between these mutations. strains carrying mutations at three of these loci have altered periods on minimal media: prd-1, several alleles at the olir (oligomycin resistance) locus and four alleles at the frq locus. a mutation at the fourth locus, cel, which results in a defect in fatty acid synthesis, also leads to ...19853155701
rna processing in neurospora crassa mitochondria: use of transfer rna sequences as signals.we have used rna gel transfer hybridization, s1 nuclease mapping and primer extension to analyze transcripts derived from several genes in neurospora crassa mitochondria. the transcripts studied include those for cytochrome oxidase subunit iii, 17s rrna and an unidentified open reading frame. in all three cases, initial transcripts are long, include trna sequences, and are subsequently processed to generate the mature rnas. we find that endpoints of the most abundant transcripts generally coinci ...19852990893
concerted evolution of dispersed neurospora crassa 5s rna genes: pattern of sequence conservation between allelic and nonallelic genes.about 100 genes coding for 5s rna in neurospora crassa are dispersed throughout the genome (selker et al., cell 24:815-818, 1981; r. l. metzenberg, j. n. stevens, e. u. selker, and e. morzycka-wroblewska, manuscript in preparation). the majority of them correspond to the most abundant species (alpha) of 5s rna found in the cell. gene conversion, gene transposition, or both may be responsible for the maintenance of sequence homogeneity (concerted evolution) of alpha-type 5s genes. to explore thes ...19852984554
[detection and intracellular localization of 2 polyphosphate phosphohydrolases with different substrate specificities in the slime variant of neurospora crassa]. 19852982559
on the role of energy metabolism in neurospora circadian clock function.neurospora crassa (bda) mycelia were kept in liquid culture. without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at ct 0-6 hr. pulses of metabolic inhibitors such as vanadate (na3vo4), molybdate (na2moo4 : 2 h2o), n-ethylmaleimide (nem), azide (nan3), cyanide (nacn) and oligomycin phase shift the circadian conidiation rhythm of neurospora crassa. maximal advance pha ...19852966687
transformation of neurospora crassa with circular and linear dna and analysis of the fate of the transforming dna.we have conducted a detailed study of 108 qa-2+ neurospora transformants which were obtained by use of circular plasmid dnas and various linear dnas. parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which the qa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass the qa-2 gene ...19852842071
molecular cloning and physical mapping of the neurospora crassa 74-or23-1a mitochondrial genome.to provide for thorough sampling of the neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the ecori digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped hincii sites. data used to locate new restriction sites were obtained from digestions of whole mitochondrial dna, digestions of the cloned ecori ...19852836095
effect of ph on the mutagenic and killing potencies of icr-170 in ad-3 tests of neurospora crassa. 20032579330
changes in gene expression elicited by amino acid limitation in neurospora crassa strains having normal or mutant cross-pathway amino acid control.the effects of amino acid limitation on gene expression have been investigated in neurospora crassa strains carrying normal (cpc-1+) or mutant (cpc-1) alleles at a locus implicated in cross-pathway amino acid control. electrophoresis and fluorography were used to reveal the patterns of label incorporation into polypeptides in vivo, or after in vitro translation of extracted mrnas. in a cpc-1+ strain at least 20% of detectable in vitro translation products showed relative increases in incorporati ...19852412092
growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of neurospora crassa.a heat-sensitive mutant of neurospora crassa, strain 4m(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. the heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group ii. the mutation defines the rip-1 gene locus. both conidial germination and mycelial extension are inhibited in the mutant at 35 degrees c and above (the nonpermissive temperature) but prolonged incubation at ...19852412091
photoregulation of some enzymes from neurospora crassa.light-grown cultures of neurospora crassa showed photoregulation of a number of enzymes. proteases and cytosolic malate dehydrogenase showed an increase in activity. there was a decrease in the activity of mitochondrial malate dehydrogenase, isocitrate dehydrogenase and cytosolic glucose-6p-dehydrogenase, isocitrate dehydrogenase and isocitrate lyase.19846239788
molecular characterization of the qa-4 gene of neurospora crassa.the qa-4 gene of neurospora crassa encodes 3-dehydroshikimate dehydratase, which catalyzes the third step of the quinic acid (qa) catabolic pathway. the enzyme has previously been purified and characterized as a monomer of approx. 37 kdal. the nucleotide sequence of the qa-4 gene is presented here and the amino acid composition and tentative nh2-terminal sequence confirm the identification of the coding region within the qa-4 dna sequence. there are no introns in the qa-4 coding region. by s1 nu ...19846241580
transcriptionally active chromatin is sensitive to neurospora crassa and s1 nucleases.we have examined the distribution of neurospora crassa and s1 nuclease cleavage products in the chromatin of the 87a7 heat shock locus of drosophila melanogaster. both of these nucleases generate single and double-strand breaks in chromatin at specific sites in the 87a7 locus. before heat induction, we find that the 5' ends of the two 87a7 hsp 70 genes contain n. crassa and s1 nuclease hypersensitive sites, while there are only a few cleavage products from elsewhere in the locus. with n. crassa ...19846096552
processing peptidase of neurospora mitochondria. two-step cleavage of imported atpase subunit 9.subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial f1f0-atpase is a nuclearly coded protein in neurospora crassa. it is synthesized on free cytoplasmic ribosomes as a larger precursor with an nh2-terminal peptide extension. the peptide extension is cleaved off after transport of the protein into the mitochondria. a processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and ...19846237909
effects of respiratory inhibitors on respiration, atp contents, and the circadian conidiation rhythm of neurospora crassa.effects of respiratory inhibitors on the circadian clock, respiratory activity, and atp content were examined in neurospora crassa. all inhibitors, potassium cyanide, sodium azide, antimycin a, and carbonyl cyanide m-chlorophenyl hydrazone (cccp), shifted the phase of the conidiation rhythm. all the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. phase shifting by azide and cccp was proportional to the lowering ...198416663893
preparation of a cell-free translation system from a wild-type strain of neurospora crassa and translation of pyruvate kinase messenger rna.a cell-free in vitro translation system exhibiting high activity has been developed from wild-type neurospora crassa mycelium. the isolation is simple and fast, and the homogenization does not appear to affect the activity of mycelial proteases and nucleases. this system is capable of supporting efficient translation of exogenously added homologous rna as demonstrated by the experiments with pk-specific mrna. in addition, it translates heterologous rna efficiently, shown by the translation of gl ...19846240997
dna-dna and dna-protein crosslinking and repair in neurospora crassa following exposure to nitrogen mustard.a new method was developed for the analysis of dna-protein crosslinks in neurospora crassa. the formations of dna-protein and dna-dna crosslinks were assayed following exposure of spheroplasts to hn2. both types of crosslink were detected and were found to be repaired during recovery. moreover a mutant sensitive to hn2 was defective in the removal of both types of crosslink.20156239978
regulation of amino acid utilization in neurospora crassa: effect of nmr-1 and ms-5 mutations.the effect of the nmr-1 and ms-5 mutations, which lead to insensitivity to glutamine-mediated nitrogen metabolite repression, was examined with respect to extracellular deaminase production by neurospora crassa. deaminase production normally requires nitrogen limitation, but these mutations eliminated this requirement and allowed production of deaminase activity under nitrogen metabolite repressing conditions. demonstration of normal glutamine transport by both strains eliminated the possibility ...19846238946
nitrogen regulation of amino acid utilization by neurospora crassa.the production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of neurospora crassa. this locus is the sole major nitrogen regulatory locus described for n. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. production of deaminase activity requires the lifting of nitrogen metabolite repression, ...19846238945
mitochondrial dna of the filamentous ascomycete cochliobolus heterostrophus : characterization of the mitochondrial chromosome and population genetics of a restriction enzyme polymorphism.the mitochondrial chromosome of cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. a physical map of c. heterostrophus mtdna was constructed using the restriction enzymes bamhi, ecori, and pvuli by dna-dna hybridizations with cloned or purified mtdna bamhi fragments. the following sequences were located on the mtdna map: (1) the large and small mitochondrial ribosomal rna genes (identified by heterologous hybridization to cloned neu ...198424178002
structure of the cell wall proteogalactomannan from neurospora crassa. ii. structural analysis of the polysaccharide part.the native proteoheteroglycan (phg) from mycelia of neurospora crassa contain two kinds of carbohydrate chains differing structure. the oligosaccharides containing mannose and galactofuranose are attached by o-glycosidic linkages to serine or threonine residues in the protein (j. biochem. 96, 1005-1011, 1984). the second kind of carbohydrate chain is a polysaccharide containing mannose and galactofuranose as the main sugar components. the results of structural studies with methylation and nmr an ...19846240491
chitosomes from the wall-less "slime" mutant of neurospora crassa.cell-free extracts from the wall-less slime mutant of neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. the yield of chitosomal chitin synthetase from slime cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption)--an indication that chitosomes are not fragments of larger membranes produced by harsh (balli ...19846240239
purification of the neurospora crassa plasma membrane h+-atpase by high-pressure liquid chromatography in the presence of sodium dodecyl sulfate.current methods for purifying the mr 100,000 h+-atpase from the plasma membrane of fungi and higher plants rely on detergent solubilization followed by density gradient centrifugation. the procedure yields catalytically active enzyme of high purity but takes several days, and the yields are low. for chemical studies on the primary structure of this enzyme, an alternative more rapid procedure was sought. in this paper a method which uses a high-performance gel filtration column in the presence of ...19846240211
two intervening sequences in the atpase subunit 6 gene of neurospora crassa. a short intron (93 base-pairs) and a long intron that is stable after excision.a 3590 base-pair region of the mitochondrial genome of neurospora crassa, including the gene for atpase subunit 6 (oli2), has been sequenced. the oli2 gene is interrupted by two intervening sequences. the first intron, situated after the third codon of the gene, is 93 base-pairs long; two-thirds of this intron consist of a palindromic sequence. the second intron is 1370 base-pairs long and contains an extended open reading frame that is continuous and in frame with the upstream exon sequence. th ...19846238172
a basal unit of valine-sensitive acetolactate synthase of neurospora crassa.valine-sensitivity as well as activity of acetolactate synthase of neurospora crassa was stabilized with 1.2 m potassium phosphate buffer during extraction from mitochondria and early stages of purification, and with 20% glycerol plus 5 mm sodium pyruvate during sephadex g200 gel chromatography. the enzyme was expressed as four molecular species having the molecular weights of about 500,000, 140,000, 68,000 and 51,000, respectively. the first and the third species showed valine-sensitivity, but ...19846237648
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