Guest: a 98 by inverted repeat transposable element in Neurospora crassa

The region immediately 3′ of histidine-3 has been cloned and sequenced from two laboratory strains of the ascomycete fungus Neurospora crassa; St Lawrence 74A and Lindegren, which have different derivations from wild collections. Amongst the differences distinguishing these sequences are insertions ranging in size from 20 to 101 by present only in St Lawrence. The largest of these is flanked by a 3 by direct repeat, has terminal inverted repeats (TIR) and shares features with several known transposable elements. At 98 bp, it may be the smallest transposable element yet found in eukaryotes. There are multiple copies of the TIR in the Neurospora genome, similar but not identical to the one sequenced. PCR amplification of Neurospora genomic DNA, using 26 by of the TIR as a single primer, gave products of discrete sizes ranging from 100 by to about 1.3 kb, suggesting that the element isolated (Guest) may be a deletion derivative of a family of larger transposable elements. Guest appears to be the first transposable element reported in fungi that is not a retrotransposon.     

Exploitation of a cellulose-binding domain from Neurospora crassa

After eight decades as a purely research organism, Neurospora crassa is becoming a production system for heterologous peptides. The present work exploits the cbh-1 gene, which encodes a class C cellobiohydrolase (EC 3.2.1.91) and has, at its carboxy-terminus, a domain with homology to other fungal cellulose-binding domains. We describe the construction of two translational fusions of the putative cellulose-binding domain with a reporter gene, which is the catalytic domain of the gla-1 glucoamylase gene of the same species, their transformation back into the organism, and expression of the constructs as cellulose-binding glucoamylase activity. This adds to the developing biotechnology of the organism the potential for enzyme/protein immobilisation.     

Sequence analysis of WIS-2-1A,a retrotransposon-like element from wheat

WIS-2-1A, a 8624 bp insertion in the Glu-1A-2 locus of chromosome 1A of wheat, consists of two 1755 bp long terminal repeats enclosing a 5114 bp internal region. No long open reading frames could be found, but inspection of the predicted amino acid sequence showed regions with homology to retrotransposon structures, including a methionine tRNA initiator binding site, a nucleotide binding domain, a protease, an integrase and a polymerase. DNA replication errors have resulted in frame-shifts in the protein coding region, suggesting that retrotransposition of WIS-2-1A, if it occurs, must be mediated by trans-acting factors.     

Characterization of a novel plasmid-like element in Neurospora crassa derived mostly from the mitochondrial DNA.

We have identified a plasmid-like element within mitochondria of Neurospora crassa strain stp-B1. It is derived from the EcoRI-4 and EcoRI-6 regions of the mitochondrial DNA, and an additional 124 bp DNA segment of unknown origin. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. The abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. An unusually large number of palindromes capable of forming secondary structures are present in the plasmid. Such a palindrome, located near sequences reminiscent of mammalian and fungal mtDNA origins of replication, may define the replication origin of the plasmid. This putative origin might also represent the replication origin of the wild-type mtDNA.     

Characterization of Ribosomes from Neurospora crassa

Ribosomes isolated from growing hyphae of Neurospora crassa contain 53 per cent protein and 47 per cent RNA and have a sedimentation coefficient of 81S at 20°C and infinite dilution. These ribosomes are stable at pH 7.4 in the presence of 0.01 M and 0.002 M MgCl₂ but undergo a dissociation into smaller particles if the MgCl₂ concentration is lowered to 0.0001 M. Two types of RNA with sedimentation coefficients of 19S₂₀⁵⁰ and 13S₂₀⁵⁰ have been extracted from the 81S particles.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

Ornithine transcarbamylase from Neurospora crassa: purification and properties

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.     

Chorismate synthase of Neurospora crassa: a flavoprotein

Chorismate synthase is purified from Neurospora crassa. The final step is accomplished by preparative electrophoresis. Its purity is estimated at ≥90% on the basis of analytical polyacrylamide gel electrophoresis. The enzyme appears to be active in at least two multimeric states, with a subunit molecular weight of ~55,000. The purified enzyme preparation is absolutely dependent on the presence of a reducing system, which can readily be provided under aerobic conditions by NADPH plus FMN or under stringent anaerobic conditions by dithionite. The following evidence implicates a physiological role for FMN in N. crassa chorismate synthase activity: (a) a preferential stimulation of activity by NADPH and FMN over other pyridine and flavin nucleotides, respectively, in both impure and purified enzyme preparations; (b) an alteration of the Chromatographic pattern of the enzyme on diethylaminoethylcellulose by the addition of FMN to the elution buffer; (c) an apparent binding of FMN to the enzyme as exhibited by gel filtration in the presence of the substrate, 3-enolpyruvylshikimate 5-phosphate; (d) a requirement for preliminary incubation with FMN, in concert with the substrate, to eliminate a reaction lag (i.e., to activate the enzyme); (e) a substrate-dependent diaphorase activity exhibited by purified enzyme preparations in the presence of FMN and NADPH. The observed activation and alteration of Chromatographic behavior of chorismate synthase by FMN suggest that the flavin nucleotide influences the conformation of the enzyme. The ability to replace NADPH and FMN with dithionite suggests that FMN mediates the flow of electrons from a source of reducing power (NADPH) to some enzymic site important to the function of the enzyme. Hence, the diaphorase activity which is observed as intrinsic to chorismate synthase of N. crassa may be significant from the standpoint of catalysis or may have importance as a regulatory mechanism.     

Purification of vacuoles from Neurospora crassa.

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

Proline oxidation by mitochondria from Neurospora crassa

Abstract Mitochondria isolated from mycelia of Neurospora crassa grown with l -proline as sole nitrogen source, oxidized l -proline at a high rate. Respiratory properties of these mitochondria and spectrophotometric measurements with artificial electron acceptors (PMS, INT) indicate that this oxidation is mediated by a flavoprotein linked to the respiratory chain.