the development of a heterologous transformation system for the cellulolytic fungus trichoderma reesei based on a pyrg-negative mutant strain.six uridine auxotroph mutants of trichoderma reesei qm 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as omp-decarboxylase negative (pyr-) by a radiometric enzyme assay. transformation to uridine prototrophy was achieved with the pyr4 gene of neurospora crassa (up to 1500 transformants/micrograms) and with pyra of aspergillus niger (700-800 transformants/micrograms). in many transformants the pyr+ function seems to be present as extrachromosomal dna. there ...19902245476
mycenon, a new metabolite from a mycena species ta 87202 (basidiomycetes) as an inhibitor of isocitrate lyase.mycenon (c11h5cl3o3), a new inhibitor of isocitrate lyase (ec was isolated from the culture broth of a basidiomycete, mycena sp. mycenon is a novel chlorinated benzoquinone derivative which is also active against bacteria and fungi. malate synthase (ec the second key enzyme of the glyoxylate cycle was not affected by mycenon. isocitrate lyase preparations from plants, bacteria and fungi were sensitive. the following ki-values for mycenon have been determined: ricinus communis, ...19902258323
expression in saccharomyces cerevisiae of a gene associated with cytoplasmic male sterility from maize: respiratory dysfunction and uncoupling of yeast mitochondria.we asked whether the mitochondrial t-urf13 gene, associated with the male sterility phenotype of t cytoplasm in maize, can be expressed in saccharomyces cerevisiae and whether this expression can mimic the effects observed in maize. we introduced the universal code equivalent of the t-urf13 gene into the s. cerevisiae nucleus by transformation and directed its translation product into mitochondria by means of a fusion with the targeting presequence from neurospora crassa at-pase subunit 9. we sh ...19902259341
an evolutionary comparison of acinetobacter calcoaceticus trpf with trpf genes of several organisms.the deduced amino acid sequence of acinetobacter calcoaceticus n-(5'-phosphoribosyl) anthranilate isomerase (prai), which is coded by trpf, was compared with trpf of caulobacter crescentus, escherichia coli, bacillus subtilis, saccharomyces cerevisiae, neurospora crassa, and aspergillus nidulans. sixty percent of identical or similar amino acids were located in alpha/beta tim (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. in addition, the analy ...19902299982
expression in yeast of the t-urf13 protein from texas male-sterile maize mitochondria confers sensitivity to methomyl and to texas-cytoplasm-specific fungal toxins.the mitochondrial gene t-urf13 from maize (zea mays l.) with texas male-sterile (t) cytoplasm codes for a unique 13 kd polypeptide, t-urf13, which is implicated in cytoplasmic male sterility and sensitivity to the insecticide methomyl and to host-specific fungal toxins produced by helminthosporium maydis race t (hmt toxin) and phyllosticta maydis (pm toxin). a chimeric gene coding for t-urf13 fused to the mitochondrial targeting peptide from the neurospora crassa atp synthase subunit 9 precursor ...19902303028
the purification and characterization of 3-dehydroquinase from streptomyces coelicolor.the enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from streptomyces coelicolor. the subunit mr estimated from polyacrylamide-gel electrophoresis in the presence of sds was 16,000. the native mr estimated by gel filtration on a superose 6 column was 209,000, indicating that the enzyme is a large oligomer. the enzyme was found to be extremely thermostable. this stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the qui ...19902306211
dna sequence analysis of the mitochondrial nd4l-nd5 gene complex from podospora anserina. duplication of the nd4l gene within its intron.a 15 kb region of the 100 kb mitochondrial genome of podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). the genes for nd4l and nd5 are identified as contiguous genes with overlapping termination and initiation codons. in race a (101 kb) the gene for nd4l (4.3 kb) has a gene duplication within an intron including a second subgroup ic intron. race s (95 kb) lacks this second gene complex. each intron has the identical 5' exon boundary. secondary structure analysis showed t ...19902319602
cloning and analysis of beta-tubulin gene from a protoctist.we have isolated and characterized by restriction endonuclease mapping, transcription pattern, and dna sequencing a beta-tubulin gene from the coenocytic freshwater protoctist, achlya klebsiana. the gene is intronless and has a single open reading frame that encodes a 444-amino acid residue polypeptide of mr 49,856. the protein shows a high degree of homology to other beta-tubulins, 85% identity to human beta-tubulin and 89% identity to beta-tubulin of the sporozoan (also a protoctist) plasmodiu ...19902394720
nuclear pre-mrna introns: analysis and comparison of intron sequences from tetrahymena thermophila and other eukaryotes.we have sequenced 14 introns from the ciliate tetrahymena thermophila and include these in an analysis of the 27 intron sequences available from seven t. thermophila protein-encoding genes. consensus 5' and 3' splice junctions were determined and found to resemble the junctions of other nuclear pre-mrna introns. unique features are noted and discussed. overall the introns have a mean a + t content of 85% (21% higher than neighbouring exons) with smaller introns tending towards a higher a + t con ...19902402440
probing fungal mitochondrial evolution with trna.sequence data are now available for almost the entire complement of mitochondrial rrnas from five fungi: schizosaccharomyces pombe, saccharomyces cerevisiae, toropulis glabrata, aspergillus nidulans and neurospora crassa. analysis of these data show that the five mitochondria can be related to a common ancestor. the unusually high similarity between some s. pombe mt trnas may be due to a process similar to gene conversion. using the number of differences between trna pairs as a measure of the ev ...19852417640
simultaneous purification of three mitochondrial enzymes. acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from neurospora crassa.the early enzymes of arginine biosynthesis in neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. the final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. we have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) w ...19862420793
h+/atp stoichiometry of proton pumps from neurospora crassa and escherichia coli.a kinetic method has been used to measure the apparent stoichiometry of h+ ions translocated per atp split by membrane-bound [h+]-atpases. in this method, membrane vesicles are suspended in well-buffered medium, atp is added, and a fluorescent probe of delta ph (acridine orange) is used to detect the formation of a steady-state ph gradient. at the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. the pump is the ...19862425739
immunological cross-reactivity of fungal and yeast plasma membrane h+-atpase.the plasma membrane h+-atpases from fungi and yeasts have similar catalytic and molecular properties. a structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kda polypeptide of the plasma membrane h+-atpase from neurospora crassa. a strong cross-reactivity is observed between the fungal h+-atpase and the enzyme from the yeasts saccharomyces cerevisiae and schizosaccharomyces pombe. structural homologies are indicated also by the ana ...19862428661
gamma-ray-sensitive mutants of neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines.well characterized gamma-ray sensitive mutants of the fungus neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (at). two neurospora mutants, uvs-6 and mus-9, show the at cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of dna synthesis following treatment with these agents. normal human or neurospora cells show an extensive biphasic dna synthesi ...19872434849
particular rna primer from growth medium differentially stimulates in vitro dna synthesis and in vivo cell growth of neurospora crassa and its slime mutant.purine rich small "rna-primer" molecules (about 10-12 nucleotides), secreted into the growth medium of 3-h germinated conidia of n. crassa, strongly stimulated a concentration-dependent in vitro dna synthesis of n. crassa slime mutant as well as dnas from the human cancer cells but did not affect that from normal cells. these "rna-primer" molecules stimulated also in vivo cell growth of n. crassa slime mutant, but not of the n. crassa wild type. our studies suggest that dnas from the slime mutan ...19872452704
purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography.following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. upon development with a 60 to 100% (v/v) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. only the lipids and very hydrophobic proteins were retained and resolved. most noticeable among retained proteins was the mr 100,000 catalytic p ...19882454596
inhibition by aluminum hydroxide of the voltage-dependent closure of the mitochondrial channel, vdac.micromolar quantities of aluminum have been found (dill et al. (1987) j. membrane biol. 99, 187-196) to reduce the voltage dependence of the mitochondrial outer membrane channel, vdac, from neurospora crassa. in the present study, various metallic and organic ions were tested for possible aluminum-like effect, and only the trivalent metals exhibited a similar ability to reduce the channels voltage dependence. however, trivalency alone was not sufficient because lanthanum (iii) had no effect. qua ...19892469483
pneumocystis carinii: sequence from ribosomal rna implies a close relationship with fungi.pneumocystis carinii is the etiologic agent of a lethal pneumonia which occurs in patients with the acquired immune deficiency syndrome (aids) and other immunocompromised hosts. the basic biochemical and genetic characteristics of p. carinii are poorly understood and its taxonomic classification as a protozoan is uncertain. to address the taxonomic question, a method was developed for the extraction of total rna from p. carinii. denaturing agarose gel electrophoresis showed the two ribosomal rna ...19892470612
pneumocystis carinii: taxing taxonomy.the detailed taxonomy of pneumocystis carinii has not been decided. most experts agree that it is either a fungus or a protozoan. in favor of a fungal designation are the recent reports that 16s and 18s rrna sequences of p. carinii have greater homology to saccharomyces cerevisiae and neurospora crassa than to selected protozoa. other studies show that sequences of the 18s and 26s rrna, microtubular ultrastructure, membrane fusion potential and dna content per cell resemble protozoa more than fu ...19892477272
characterization of the mitochondrial porin from drosophila melanogaster.mitochondrial porin was isolated from the fruit fly drosophila melanogaster at different developmental stages, starting from whole mitochondria. the porin from adults' mitochondria was fully characterized. the protein had a molecular mass of 31 kda as judged from sodium dodecylsulfate electrophoretograms. it was very resistive against digestion with v8 proteinase of staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. drosophila porin showed litt ...19892480813
dna sequence and secondary structures of the large subunit rrna coding regions and its two class i introns of mitochondrial dna from podospora anserina.dna sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal rna (rrna) from podospora anserina is interrupted by two class i introns. the coding region for the large subunit rrna itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. secondary structure models for the large subunit rrna were constructed and compared with the equivalent structure from escherichia coli 23s rrna. the two structures were remarkably similar despite ...19892494353
genetic transformation system for the aflatoxin-producing fungus aspergillus flavus.a heterologous transformation system was developed for aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of dna. protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of neurospora crassa. transformants were selected for their ability to grow and sporulate on medium lacking uracil. vector dna appeared to integrate randomly into the genome of a. flavus with a tendency for multiple, ta ...19892495764
[properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in pichia guilliermondii yeasts].2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (nh4)2so4 at 45-75% of saturation and chromatography on blue sepharose cl-6b. the use of gel filtration through sephadex g-150 and chromatography on deae-cellulose proved to be less effective for the enzyme purification. it has been established that it is 2,5-diamino-4-o ...19892511652
a mitochondrial protein from neurospora crassa detected both on ribosomes and in membrane fractions. analysis of the gene, the message, and the protein.we have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from neurospora crassa by screening a lambda gt11 cdna library with an antiserum against a mixture of these proteins. the cdna and genomic dna sequence for one of these genes, mrp-3, was determined. the mrp3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospect ...19892521217
nucleotide sequence of the neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in saccharomyces cerevisiae and escherichia coli.the complete nucleotide sequence of the neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. a comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in saccharomyces cerevisiae (encoded by the trp5 gene) and escherichia coli (encoded by the trpa and trpb genes) shows that the a and b domains (amino acid segments homologous to the trpa and trpb polypeptides, respectively) of the n. crassa and y ...19892521855
molybdenum cofactor biosynthesis in humans. identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor.molybdenum cofactor deficiency is a devastating disease with affected patients displaying the symptoms of a combined deficiency of sulfite oxidase and xanthine dehydrogenase. because of the extreme lability of the isolated, functional molybdenum cofactor, direct cofactor replacement therapy is not feasible, and a search for stable biosynthetic intermediates was undertaken. from studies of cocultured fibroblasts from affected individuals, two complementation groups were identified. coculture of g ...19892522104
x-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. iii. genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests.genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions was made by means of complementation tests on all possible pairwise combinations of 50 x-ray-induced irreparable adenine-3 mutants (designated ad-3ir). all mutants were induced in either heterokaryon 11 or heterokaryon 12 of neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3a and ad-3b and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad ...19892522164
assembly kinetics and identification of precursor proteins of complex i from neurospora crassa.complex i from neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. several subunits were isolated. polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo. n. crassa cells were pulse-labelled with radioactive amino acids. the time course of incorporation of radioactivity into complex-i polypeptides was studied b ...19892523803
a family of mitochondrial proteins involved in bioenergetics and biogenesis.the respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. the functions of the subunits that do not contain prosthetic groups are largely unknown. the cytochrome reductase complex of neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins i and ii (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid ...19892524007
purification and characterisation of nad-glutamate dehydrogenase from aspergillus nidulans.nad-glutamate dehydrogenase has been purified from mycelia of a. nidulans. the enzyme comprises subunits of 110 kda. it is located in the cytosol. it is completely denatured by 1.0 m guanidine hydrochloride, and is not renatured by subsequent dilution. isophthalate is a strong competitive inhibitor and the enzyme is also inhibited by thiol reagents. the properties of the enzyme were compared to those from other fungi in terms of size, sensitivity to inhibitors, intracellular distribution and mod ...19892524418
recombination and replication of plasmid-like derivatives of a short section of the mitochondrial chromosome of neurospora crassa.the 21-kbp mitochondrial chromosome of the stp-ruv strain of neurospora crassa undergoes regional amplification yielding plasmid-like supercoiled circles varying in size from subunit length to very high multimers. a comparison of the base sequence of the five plasmids studied, with the region of the chromosome from which they were derived, indicated that the amplified chromosomal segments were determined by a recombination-excision process near or within two structurally distinctive regions. one ...19892524421
cys-3, the positive-acting sulfur regulatory gene of neurospora crassa, encodes a protein with a putative leucine zipper dna-binding element.the sulfur-regulatory circuit of neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. the positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. expression of the cys-3 regulatory gene was found to be controlled by ...19892524646
molecular cloning of a neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.the albino-3 (al-3) gene of neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. the n. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing n. crassa dna inserts, and the transformants were screened for the al-3+ phenotype. one al-3+ qa-2+ transformant (al3-1) was examined in detail and shown to contain intact vector sequences integrated into the n. crassa genome. the vector a ...19892524647
photoaffinity labelling of mitochondrial nadh: ubiquinone reductase with pethidine analogues.1. chemically reactive derivatives of pethidine analogues--novel potent inhibitors of the mitochondrial nadh: ubiquinone reductase (complex i)--were synthesized. 2. dose-response curves of these components revealed that the photoactivatable aryl azido derivative has retained most of the inhibitory activity displayed by the parent substance. after introduction of a radioactive iodine isotope into the molecule, it was used as a probe for the localization of the inhibitor binding polypeptides withi ...19892525381
transformation of neurospora crassa by an integrative transforming plasmid is not enhanced by ribosomal dna sequences.two integrative transforming plasmids of neurospora crassa that differed only by the presence of almost all of a ribosomal dna repeat unit on one plasmid were constructed. the plasmids were used to test the target concentration hypothesis which states that the transformation frequency is proportional to the number of genomic copies of a homologous sequence located on the transforming plasmid. since there are approx. 200 copies of the rdna sequences in the genome, the target concentration hypothe ...19892525404
an optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein.a procedure for successful analysis of the hydrophobic tryptic peptides of the neurospora crassa plasma membrane h+-atpase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) is described. the features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with sds at 0 degrees c, (iii) sds-page of the hydrophobic p ...19892525882
development of an in vitro transcription system for neurospora crassa mitochondrial dna and identification of transcription initiation sites.we have developed an in vitro transcription system for neurospora crassa mitochondrial dna (mtdna) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rrna and cytochrome b (cob). the in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-ttagara(t/g)g(t/g) ...19892528684
a unique low frequency raman band associated with metal binding to metallothionein.we find that the low frequency raman spectrum of zn(ii) metallothionein has a single prominent band at 138 cm-1 which is absent from the raman spectrum of the metal-free protein. this feature is also found for cd(ii) binding to both of the independent metallothionein domains and the metallothionein from neurospora crassa. tco(iii) coordination to metallothionein results in a similar raman band which is also found for the complex (ph4as)[reo(sch2ch2s)2]. by comparing these results to literature d ...19892528950
translocation arrest by reversible folding of a precursor protein imported into mitochondria. a means to quantitate translocation contact sites.passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the nh2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. isolated mitochondria of neurospora crassa readily imported the fusion protein. in the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accum ...19892529262
x-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa. iv. irreparable mutants of genotype ad-3a and ad-3b result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations.the induction of specific-locus mutations in the ad-3 region of neurospora crassa after x-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designated ad-3r) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3ir), and (2) the induction kinetics of each class (webber and de serres, 1965). more extensive genetic tests made subsequently (de serres, 1989a) on the 832 x-ray-induced spec ...19892529438
oxidation of neurospora crassa nadp-specific glutamate dehydrogenase by activated oxygen species.the glutamine synthetase and the nadp-specific glutamate dehydrogenase activities of neurospora crassa were lost in a culture without carbon source only when in the presence of air. glutamine synthetase was previously reported to be liable to in vitro and in vivo inactivation by activated oxygen species. here we report that nadp-specific glutamate dehydrogenase was remarkably stable in the presence of activated oxygen species but was rendered susceptible to oxidative inactivation when chelated i ...19892530208
stereoselective arginine binding is a phylogenetically conserved property of group i self-splicing rnas.we have examined the reaction of gtp with rna polymerase transcripts containing the self-splicing rna precursors from the neurospora crassa cob1 intron, and from introns in the suny, nrdb and td genes of bacteriophage t4. in each case, we find a low km for gtp (between 0.8 and 11 microm), accompanied by competitive inhibition of the gtp reaction by l-arginine, as was found for the previously examined tetrahymena nuclear pre-rrna intron. trials with the 20 standard amino acids show that inhibitio ...19892531082
molecular cloning and expression in saccharomyces cerevisiae and neurospora crassa of the invertase gene from neurospora crassa.a plasmid (named pcn2) carrying a 7.6 kb bamhi dna insert was isolated from a neurospora crassa genomic library raised in the yeast vector yrp7. saccharomyces cerevisiae suco and n. crassa inv strains transformed with pnc2 were able to grow on sucrose-based media and expressed invertase activity. saccharomyces cerevisiae suco (pnc2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type n. crassa, although s. cerevisiae suc+ did not. the cloned dna ...19892531138
sensitivity to cyclosporin a is mediated by cyclophilin in neurospora crassa and saccharomyces cerevisiae.cyclosporin a, a cyclic fungal undecapeptide produced by tolypocladium inflatum, is a potent immunosuppressive drug originally isolated as an antifungal antibiotic. cyclosporin a (csa) is widely used in humans to prevent rejection of transplanted organs such as kidney, heart, bone marrow and liver. the biochemical basis of csa action is not known: its primary cellular target has been suggested to be calmodulin, the prolactin receptor or cyclophilin, a csa-binding protein originally isolated from ...19892531848
regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-trna synthetases of neurospora crassa.we show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-trna synthetase (leurs) of neurospora crassa are distinct in their encoded proteins, codon usage, mrna levels, and regulation. the 4.2-kilobase-pair region representing the structural gene for cytoplasmic leurs and flanking regions has been sequenced. the positions of the 5' and 3' ends of mrna and of a single 62-base-pair intron have been mapped. the methionine-initiated open reading frame encoded a protein of 1,123 ami ...19892532300
bialaphos resistance as a dominant selectable marker in neurospora crassa.conidia of neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/l in westergaard's or fries' minimal media. plasmid pja4 was constructed by inserting a truncated bar gene from streptomyces hygroscopicus fused to the his-3 promoter from n. crassa into puc19. the bar gene in plasmid pja4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. the bar gene can be used as an additional dominant selectable marker for transfor ...19892532965
evidence for three differentially regulated catalase genes in neurospora crassa: effects of oxidative stress, heat shock, and development.genetic and biochemical studies demonstrated that neurospora crassa possesses three catalases encoded by three separate structural genes. the specific activities of the three enzymes varied in response to superoxide-mediated stress, heat shock, and development. the three loci, which we designated cat-1, cat-2, and cat-3, map to the right arms of chromosomes iii, vii, and iii, respectively. the cat-1-encoded enzyme (designated cat-1; estimated molecular weight, 315,000; pi 5.2) was the predominan ...19892540152
cytochrome oxidase subunit v gene of neurospora crassa: dna sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit v.the sequences of cdna and genomic dna clones for neurospora cytochrome oxidase subunit v show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid n-terminal extension. the subunit v protein sequence is 34% identical to that of saccharomyces cerevisiae subunit v; these proteins, as well as the corresponding bovine subunit, subunit iv, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. the neurospora crassa subunit v g ...19892540423
amino acid sequence of the alpha and beta subunits of methanosarcina barkeri atpase deduced from cloned genes. similarity to subunits of eukaryotic vacuolar and f0f1-atpases.the atpa and atpb genes coding for the alpha and beta subunits, respectively, of membrane atpase were cloned from a methanogen methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. the methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar h+-atpases; 52% of the residues of the methanogenic alpha subunit were identical ...19892544575
nucleotide sequence and regulation of expression of the aspergillus nidulans gdha gene encoding nadp dependent glutamate dehydrogenase.the nucleotide sequence of the aspergillus nidulans gdha gene encoding nadp linked glutamate dehydrogenase has been determined and northern blot analysis used to study the regulation of expression of this gene. the gdha gene is 1485 nucleotides long and, by comparison with the corresponding neurospora crassa am gene, has two putative introns of 53 nucleotides and a protein encoding region of 1380 nucleotides that codes for an inferred protein of 49.63 kda which shows regions of homology with glu ...19892550758
reconstitution of a light-stimulated adenylate cyclase from retina and neurospora crassa preparations. characterization of the heterologous systems using normal and degenerative retinas.adenylate cyclase catalytic subunits from neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with n-ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing mgatp. maximal activation was observed at 550 nm. transducin-depleted retinal membranes were not capable of reconstituting the heterologous light-stimulated adenylate cyclase system. ...19892553402
[identification of human porins. ii. characterization and primary structure of a 31-lda porin from human b lymphocytes (porin 31hl)].we characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kda derived from the plasmalemm of b-lymphocytes (porin 31hl). porin 31hl is shown to be a basic, channel forming membrane protein. the protein chain is composed of 282 amino acids with a relative molecular mass of 30641 da without derivatisation. it is not a glycoprotein. the n-terminus is acetylated. altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids ...19892559745
cloning and analysis of the neurospora crassa gene for cytochrome c heme lyase.the cyt-2-1 mutant of neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (mitchell, m.b., mitchell, h.k., and tissieres, a. (1953) proc. natl. acad. sci. u.s.a. 39, 606-613; nargang, f.e., drygas, m.e., kwong, p.l., nicholson, d.w., and neupert, w. (1988) j. biol. chem. 263, 9388-9394). by rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a n. crassa genomic library using sib selection. analysis of the dna ...19892572587
transformation by integration in podospora anserina. iii. replacement of a chromosome segment by a two-step process.we have developed in podospora anserina a two-step procedure for dna sequence replacement through transformation which might be applicable to other filamentous fungi. targeting of transforming dnas to their homologous locus is achieved provided a cosmid vector is used. southern blot analysis of genomic dnas from a set of transformants is presented. the data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by ...19892575706
heterogeneity of 5s rna in fungal ribosomes.neurospora crassa has at least seven types of 5s rna genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. a high resolution gel electrophoresis system was developed to separate minor 5s rna's from the major 5s rna (alpha). a study of several neurospora crassa strains, four other species in the genus neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5s rna heterogeneity is common among fungi. in addition, di ...19852579431
transformation in fungi.transformation with exogenous deoxyribonucleic acid (dna) now appears to be possible with all fungal species, or at least all that can be grown in culture. this field of research is at present dominated by saccharomyces cerevisiae and two filamentous members of the class ascomycetes, aspergillus nidulans and neurospora crassa, with substantial contributions also from fission yeast (schizosaccharomyces pombe) and another filamentous member of the class ascomycetes, podospora anserina. however, tr ...19892651864
two proteins encoded at the chla locus constitute the converting factor of escherichia coli chla1.molybdopterin (mpt) is not produced by the escherichia coli mutants chla1, chlm, or chln or by the neurospora crassa mutant nit-1. extracts of e. coli chla1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of n. crassa nit-1 into molybdopterin in vitro. in this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kda]) which can ...19892656653
acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms.proteins antigenically similar to the acyl carrier protein (acp) found in the mitochondria of neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. these mitochondrial proteins were similar to neurospora acp both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. authentic acp(s) show this type of change upon conversion of the acylated to the unacylated form. purifi ...19892680483
yeast cyclophilin: isolation and characterization of the protein, cdna and gene.cyclophilin (cph) has been isolated from the yeast saccharomyces cerevisiae, purified to homogeneity and partially sequenced. oligodeoxyribonucleotides deduced from this sequence were used to isolate the corresponding cdna and gene. an open reading frame coding for a 162-amino acid (aa) protein with a calculated mr of 17,392, was deduced from the nucleotide sequence. comparison between yeast and human cph shows a very high overall sequence conservation (65% aa homology). the binding of yeast cph ...19892687115
improved transformation efficiency of aspergillus niger using the homologous niad gene for nitrate reductase.aspergillus niger transformation frequencies of up to 1,176 transformants per micrograms dna were achieved using the plasmid vector psta10 containing the a. niger nitrate reductase structural gene. analysis of genomic endonuclease cleaved dna from nitrate utilising transformants by dna hybridisation, showed that most integration events are as a result of homologous recombination. the niad transformation system was used successfully for the introduction of the unselected escherichia coli fusion g ...19892791035
molecular characterization of trp1, a gene coding for tryptophan synthetase in the basidiomycete coprinus cinereus.we utilized a cloned gene (trp5) encoding tryptophan synthetase (tsase) from saccharomyces cerevisiae to identify and clone the corresponding gene (trp1) from the basidiomycete coprinus cinereus. the primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for tsase. a transformation assay was used to demonstrate that 321 nt, which do not include caat or tataaa elements and precede the translation initia ...19892806911
the molybdenum iron-sulphur protein from desulfovibrio gigas as a form of aldehyde oxidase.the molybdenum iron-sulphur protein originally isolated from desulfovibrio gigas by moura, xavier, bruschi, le gall, hall & cammack [(1976) biochem. biophys. res. commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(v). the signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 hgz in 1h2o and 2h2o and computer simulations, to have parameters corresponding to those of the slow signal from the inactive ...19872821990
purification and characterization of an endo-exonuclease from saccharomyces cerevisiae that is influenced by the rad52 endo-exonuclease has been purified from logarithmically growing cells of the yeast saccharomyces cerevisiae. identification and purification of this nuclease was facilitated by its being precipitable with an antibody raised against a previously described neurospora crassa endo-exonuclease (resnick, m. a., chow, t. y.-k. nitiss, j., and game, j. c. (1984) cold spring harbor symp. quant. biol. 49, 639-649 and t. y.-k. chow and m. a. resnick (1988) mol. gen. genet., in press). the enzyme which w ...19872826428
nitrobacter winogradskyi cytochrome a1c1 is an iron-sulfur molybdoenzyme having hemes a and c.cytochrome a1c1 (nitrite-cytochrome c oxidoreductase) purified from nitrobacter winogradskyi (formerly n. agilis) contained molybdenum, non-heme iron, and acid-labile sulfur in addition to hemes a and c; it contained 1 mol of heme a, 4-5 g atoms of non-heme iron, 2-5 g atoms of acid-labile sulfur, and 1-2 g atoms of molybdenum per mol of heme c, but did not contain copper. the fluorescence spectra of the molybdenum cofactor derivative prepared from cytochrome a1c1 were very similar to those of t ...19872828343
isolation and analysis of the neurospora crassa cyt-21 gene. a nuclear gene encoding a mitochondrial ribosomal protein.the neurospora crassa nuclear mutant cyt-21-1 (originally 297-24; pittenger, t.h., and west, d.j. (1979) genetics 93, 539-555) has a defect leading to gross deficiency of mitochondrial small ribosomal subunits. here, we have cloned the cyt-21+ gene from a n. crassa genomic library, using the sib selection procedure (akins, r. a., and lambowitz, a. m. (1985) mol. cell biol. 5, 2272-2278). the genomic clone contains a short split gene encoding a basic protein of 107 amino acid residues. this prote ...19882830266
a 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in neurospora crassa.we showed previously that the cyt-21+ gene of neurospora crassa encodes a mitochondrial ribosomal protein homologous to escherichia coli ribosomal protein s-16 (kuiper, m. t. r., akins, r. a., holtrop, m., de vries, h., and lambowitz, a. m. (1988) j. biol. chem. 263, 2840-2847). a mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rrna (collins, r. a., bertrand, h., lapolla, r. j., and lambowitz, a. m. (1979) mol. gen. genet. 177, 73-84). i ...19882830267
an endo-exonuclease activity of yeast that requires a functional rad52 gene.extracts of rad+ and radiation-sensitive (rad) mutants of the yeast saccharomyces cerevisiae were examined for total mg2+-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endo-exonuclease from neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. no significant differences were observed in total deoxyribonuclease activity between rad+ and rad mutants. the antibody preci ...19882830467
inversions and recombinations in mitochondrial dna of the (sg-1) cytoplasmic mutant in two neurospora species.the mitochondrial dnas of [sg-1] cytoplasmically-mutant and wild-type strains of neurospora crassa and neurospora sitophila were examined by comparative restriction endonuclease analyses. the mtdna of n. sitophila wild type of whitehouse differs from type ii mtdna of n. crassa by insertions of 3.3 kb in ecori-9, and 1.2 kb in ecori-3, and a deletion of 1.1 kb in ecori-5. these dna heteromorphisms provided convenient markers for tracing n. crassa [sg-1] mtdna during and after its transfer into n. ...19862832078
isolation and identification of the aspergillus nidulans gdha gene encoding nadp-linked glutamate dehydrogenase.the neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed aspergillus nidulans gene libraries. these clones have a common hindiii 1.85 kb fragment. this a. nidulans nucleotide stretch hybridises to a n. crassa 2.7 kb bamhi fragment of wild type dna but not to a co-migrating fragment from the dna of the n. crassa am132 deletion mutant. one a. nidulans clone was shown to complement the n. crasse am132 deletion strain. the n. crassa transfo ...19862834076
identification and isolation of a putative permease gene in the quinic acid utilization (qut) gene cluster of aspergillus nidulans.mutations in the qutd gene of aspergillus nidulans cause the loss of ability to grow upon quinic acid as sole carbon source in media at normal ph 6.5 and failure to induce three enzyme activities specifically required for metabolism to protochatechuic acid. all 9 qutd mutants recovered are recessive and have been found to be ph sensitive, growing strongly on quinic acid media at ph 3.5 and producing significant induced enzyme activities. these properties are consistent with the hypothesis that t ...19872835177
neurospora crassa nuclear genome contains analogy of saccharomyces cerevisiae genes for ribosomal rna processing.neurospora crassa wild type genome shows dna sequences which are homologous to the sequences present in the rrna processing genes of the yeast saccharomyces cerevisiae. five such processing genes from yeast, viz., rna1 through rna5, cloned in plasmid pbr322 were transformed in escherichia coli strain le392. southern blots containing dnas from these clones were restricted with several restriction endonucleases along with dnas from lambda phage, rice (plant) and neuroblastoma (animal), were hybrid ...19872835180
sequence analysis of the ddpyr5-6 gene coding for ump synthase in dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and omp decarboxylases.a dictyostelium discoideum dna fragment that complements the ura3 and the ura5 mutants of saccharomyces cerevisiae has been sequenced. it contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. this gene, named ddpyr5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (oprtase) and the orotidine-5'-phosphate decarboxylase (ompdecase) domains described for ump synthase in mammals. the existence of separate domains ...19882835631
transformation of aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene.a homologous transformation system for the filamentous fungus aspergillus niger has been developed, based on the orotidine-5'-phosphate-decarboxylase gene. a. niger pyr- mutants have been selected from 5-fluoro-orotic acid resistant mutants. these mutants were found to comprise two complementation groups, pyra and pyrb. the a. niger omp-decarboxylase gene was isolated from a gene library by heterologous hybridization with the neurospora crassa pyr4 gene. the cloned gene is capable to transform a ...19872836081
nucleotide sequence of the aspergillus niger trpc gene: structural relationship with analogous genes of other organisms.the nucleotide sequence of the aspergillus niger tryptophan c (trpc) gene was determined. northern hybridization and s1-mapping experiments showed the presence of a 2.6 kb trpc poly(a)+ rna with two very short (5 and 6 nucleotides) noncoding 5'-regions. comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (g, c, f) in the a. niger trpc protein which catalyse the glutamine amidotransferase reaction (gat), ...19882836085
nucleotide sequence of the mitochondrial cytochrome oxidase subunit ii gene in the yeast hansenula saturnus.the gene for subunit ii of cytochrome oxidase in the yeast hansenula saturnus was previously shown to be located on a 1.7 kb hindiii-bamhi fragment of mitochondrial dna (lawson and deters, accompanying paper). in this paper, we report the nucleotide sequence of a large part of this fragment, covering the coding region of the subunit ii gene, designated coxii, and its 5' and 3' flanking regions. the coding region of the coxii gene consists of a continuous open reading frame, 744 nucleotides long, ...19852836090
dnas of the two mating-type alleles of neurospora crassa are highly dissimilar.the mating-type alleles a and a of neurospora crassa control mating in the sexual cycle and function in establishing heterokaryon incompatibility in the vegetative cycle. the a and a alleles were cloned, and they were shown to encode both the sexual functions and vegetative incompatibility. the mating-type clones contain nonhomologous dna segments that are flanked by common dna sequences. neurospora crassa and all heterothallic and pseudohomothallic neurospora species contain a single copy of on ...19882840740
organization of the ribosomal rna genes of schizophyllum commune.the 18, 5.8, 25 and 5s ribosomal rna (rrna) cistrons have been mapped on the ribosomal dna (rdna) unit repeat of schizophyllum commune strain 4-40. these genes are spatially ordered in the sequence given. the presence of a large primary precursor rrna which is processed to form the mature 18, 5.8 and 25s rrnas has been demonstrated. we have mapped the site of transcriptional initiation for this rrna primary precursor. the sequence surrounding this site has been determined and shown to be highly ...19882841031
informational parameters and randomness of mitochondrial dna.the informational content of genomes of nuclear and mitochondrial origin is examined. by using the parameters of shannon's information theory the language of mitochondrial dna is shown to be more similar to the language of bacterial dna than to that of nuclear dna in more evolutionarily advanced animals. moreover, using the parameters of kolmogorov's theory on randomness, genes of different organisms (neurospora crassa and saccharomyces cerevisiae) coding for the same protein (subunit 9 of atpas ...19882842512
the isolation of demolybdo xanthine oxidase from bovine was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. this has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described nishino & tsushima [(1986) j. biol. chem. 261, 11242-11246]. enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. like the normal e ...19882850803
functional effects and cross-reactivity of antibody to purified subunit b (uncf protein) of escherichia coli proton-atpase.subunit b (uncf protein) of the proton-atpase (f1f0) of escherichia coli was purified from membranes of strain an1460 (unc+). antibody to purified subunit b was raised in rabbits. it reacted with f1-depleted membranes and blocked f1 binding. bound antibody had no effect on proton transport through f0. f1-depleted membranes competed with purified subunit b for antibody in an enzyme-linked immunosorbent assay. f1-depleted membranes which had been pretreated with trypsin or preincubated with satura ...19852857549
size of the plasma membrane h+-atpase from neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum ca2+-atpase from skeletal muscle.using radiation inactivation, we have measured the size of the h+-atpase in neurospora crassa plasma membranes. membranes were exposed to either high energy electrons from a van de graaff generator or to gamma irradiation from 60co. both forms of radiation caused an exponential loss of atpase activity in parallel with the physical destruction of the mr = 104,000 polypeptide of which this enzyme is composed. by applying target theory, the size of the h+-atpase in situ was found to be approximatel ...19852862141
labeling of individual amino acid residues in the membrane-embedded f0 part of the f1 f0 atp synthase from neurospora crassa. influence of oligomycin and dicyclohexylcarbodiimide.three f0 subunits and the f1 subunit beta of the atp synthase from neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125i]iodophenyl)diazirine ([125i]tid). in the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the nh2-terminus and five residues at the c-terminus of the protein. labeling occurred at similar positions compared with the homologous protein (subunit c) in the atp synthase ...19862869944
topological studies suggest that the pathway of the protons through f0 is provided by amino acid residues accessible from the lipid phase.the structure of the f0 part of atp synthases from e. coli and neurospora crassa was analyzed by hydrophobic surface labeling with [125i]tid. in the e. coli f0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. labeled amino acid residues were identified by edman degradation of the dicyclohexylcarbodiimide binding (dccd) proteins from e. coli and neurospora crassa. the very similar patterns obtained with the two ...19862874840
primary structure of the neurospora plasma membrane h+-atpase deduced from the gene sequence. homology to na+/k+-, ca2+-, and k+-atpase.the gene for the neurospora crassa plasma membrane h+-atpase has been cloned and sequenced. the gene encodes for a protein of 920 amino acids with a molecular weight of 100,002. the coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus. the deduced amino acid sequence of the n. crassa plasma membrane h+-atpase exhibits 75% homology to the amino acid sequence of the saccharomyces cerevisiae plasma membrane h+-atpase. also, an amino acid com ...19862876992
the atp synthase subunit 9 gene of aspergillus nidulans: sequence and transcription.we have determined the nucleotide sequence of the aspergillus nidulans nuclear gene olic31, which encodes subunit 9 of mitochondrial atp synthase. the open reading frame contains no introns and specifies a predicted protein of 143 amino acids comprising a pre-sequence of 62 residues and a mature protein of 81 residues. the amino acid homology with the equivalent neurospora crassa protein is 50% for the pre-sequence and 80% for the mature protein. a comparison with this and other imported mitocho ...19862880279
yeast mitochondrial atpase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle.subunit 8 of yeast mitochondrial f1f0-atpase is a proteolipid made on mitochondrial ribosomes and inserted directly into the inner membrane for assembly with the other f0 membrane-sector components. we have investigated the possibility of expressing this extremely hydrophobic, mitochondrially encoded protein outside the organelle and directing its import back into mitochondria using a suitable n-terminal targeting presequence. this report describes the successful import in vitro of atpase subuni ...19862881782
human liver cdna clones encoding proteolipid subunit 9 of the mitochondrial atpase complex.clones encoding the proteolipid subunit 9 of the mitochondrial atpase complex have been isolated from a lambda gt10 library of human liver cdna sequences, using a probe of neurospora crassa cdna encoding subunit 9. from nucleotide sequence analysis it is concluded that the amino acid sequence of mature human subunit 9 is identical to that of its bovine counterpart. by comparing the sequence of two cdna clones (denoted human 1 and 2) to those of two bovine cdna clones (denoted p1 and p2) recently ...19872883974
studies on nitrate reductase of clostridium perfringens. iv. identification of metals, molybdenum cofactor, and iron-sulfur cluster.nitrate reductase of clostridium perfringens was purified by an improved method using immuno-affinity chromatography. the purified preparation contained mo, fe, and acid-labile sulfide; the mo content was 1 mol per mol and the fe 3.7 mol per mol of the enzyme. the inactive enzyme obtained from cells grown in the presence of tungstate did not hold mo but contained 1 mol of w. the content of fe was not increased. the presence of molybdenum cofactor in the nitrate reductase was indicated by the for ...19872884214
mutation of a conserved glycine residue modifies the vanadate sensitivity of the plasma membrane h+-atpase from schizosaccharomyces pombe.the structural gene pma+1 for the h+-atpase from the fission yeast schizosaccharomyces pombe has been isolated and sequenced. the intron-less gene encodes for a protein of mr = 99,769 which is 75% homologous to those of saccharomyces cerevisiae and neurospora crassa. the s. pombe pma+1 gene complements not only s. pombe pma-1-1 but also s. cerevisiae pma-1-4 mutants selected for in vitro vanadate-resistant atpase activity. the sequence of the s. pombe mutant pma-1-1 allele reveals that the glyci ...19872891694
clathrin coated vesicles in neurospora crassa.electropherograms of neurospora crassa homogenates showed a polypeptide with a mobility slightly lower than that of a standard sample of clathrin (from bovine brain). subcellular fractionation of the homogenate resulted in a 20-fold enrichment of the putative n. crassa clathrin in the microsomal fraction. further fractionation of the microsomal fraction by glass bead permeation chromatography yielded a fraction enriched about 150-fold relative to the homogenate. coated vesicles (42.5 +/- 2.5 nm ...19872892127
assembly of functional proton-translocating atpase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.a mitochondrial gene from saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the f0/f1-type mitochondrial atpase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. the experiments reported here utilized a multicopy expression vector (plf1) that replicates in the nucleus of yeast cells and that carries an inserted dna segment, specifying a precursor protein (n9/y8) consisting of subunit 8 fused to an n- ...19882895470
reprogrammed expression of subunit 9 of the mitochondrial atpase complex of saccharomyces cerevisiae. expression in vitro from a chemically synthesized gene and import into isolated mitochondria.a synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial atpase complex. this gene (nap9) incorporates codons frequently used in nuclear genes of saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product. follow ...19882895707
studies on the import into mitochondria of yeast atp synthase subunits 8 and 9 encoded by artificial nuclear fusions have been constructed between each of subunits 8 and 9 from mitochondrial atpase of saccharomyces cerevisiae, proteins normally encoded inside mitochondria, and the cleavable n-terminal transit peptide from the nuclearly encoded precursor to subunit 9 of neurospora crassa mitochondrial atpase. the subunit 8 construct was imported efficiently into isolated yeast mitochondria and was processed at or very near the fusion point. when expressed in vivo from its artificial nuclear gene, ...19882900779
biosynthesis of the plasma membrane h+-atpase of neurospora crassa.the plasma membrane h+-atpase of neurospora is a 100-kda integral membrane protein which appears, on the basis of hydropathy analysis of its amino acid sequence, to span the lipid bilayer at least eight times. to investigate the assembly and processing of the atpase, a full-length cdna has been constructed for use in in vitro transcription and translation experiments. comparison of three different forms of the atpase (nascent protein, nascent protein cotranslationally inserted into membranes, an ...19882902084
primary structure of cucumber (cucumis sativus) ascorbate oxidase deduced from cdna sequence: homology with blue copper proteins and tissue-specific expression.cdna clones for ascorbate oxidase were isolated from a cdna library made from cucumber (cucumis sativus) fruit mrna. the library was screened with synthetic oligonucleotides that encode the nh2-terminal sequence of this enzyme. nucleotide sequence analysis of the cloned cdna inserts revealed a 1761-base-pair open reading frame that encoded an nh2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (mr, 62,258). the amino acid sequence deduced from nucleotide sequence ...19892919172
nucleotide sequence of the gdh gene coding for the nadp-specific glutamate dehydrogenase of saccharomyces cerevisiae.the isolation of the saccharomyces cerevisiae gene for nadp-dependent glutamate dehydrogenase (nadp-gdh) by cross hybridization to the neurospora crassa am gene, known to encode for nadp-gdh is described. two dna fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. a yeast shuttle vector (cv13) carrying either to the cloned fragments complements the gdh- strain of s. cerevisiae and directs substantial overprod ...19852932370
carbamoyl-phosphate synthetases from neurospora crassa. immunological relatedness of the enzymes from neurospora, bacteria, yeast, and mammals.neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (cps-a) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. we have prepared antiserum against a highly purified preparation of the large subunit of cps-a and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight prec ...19852932445
isolation and characterization of the aspergillus niger trpc gene.the aspergillus niger trpc gene was isolated by complementation experiments with an escherichia coli trpc mutant. plasmid dna containing the a. niger trpc gene transforms an aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpc gene, to trp+, indicating the presence of a complete and functional trpc gene. southern blot analysis of dna from these trp+ transformants showed that plasmid dna was present but that this dna was not integrated at the site of the chr ...19852936650
neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein.when neurospora crassa was labeled with [14c]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. the 19-kda band was converted to the 22-kda band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral ph, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral p ...19862937780
a study of the messenger rna encoding pyruvate kinase of neurospora neurospora crassa, there is a single pyruvate kinase (pk) consisting of four identical subunits of approximately 60k daltons. northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mrna species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dt)cellulose chromatography. these messages are present in polysomes, immuno-precipitated by anti-pk antibodies, indicati ...19862941081
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