Expression of a plant protein by Neurospora crassa

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.     

Repressible cation-phosphate symporters in Neurospora crassa

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

A putative rhamnogalacturonase required for sexual development of Neurospora crassa

In previous work, the asd-I (ascus development) gene of the filamentous fingus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-I gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-I protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-I crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-I asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-I protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

A suppressor mutation which suppresses adenylyl cyclase mutations in Neurospora crassa

Membranous obstruction of the inferior vena cava at the suprahepatic level is rare. The present report summarizes direct operative repair. A review of alternative therapeutic strategies is presented.     

Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount f growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive temperature. Incubation of the mcb mutant at restrictive temperature results in a three- to fivefold increase in the level of extracellular protein and a 20-fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-1 double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced after a shift of the mcb mutant to restrictive temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.     

Energy and metabolic intercellular interaction in the growth region of Neurospora crassa

A major group of systemic autoimmune diseases is associated with abnormal lymphoproliferation, as a result of defects in the termination of lymphocyte activation and growth. Recent progress has been made in understanding the causes and consequences of these abnormalities. At the molecular level, the defects in CD95 and its ligand are only the most obvious reasons for the breakdown of 'clonal contraction' which in fact requires the participation of multiple gene products, including the IL-2-IL-2-receptor system, to set up a functional apoptotic machinery.     

Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia

We have previously demonstrated that blue light induces the phosphorylation of a 15-kDa protein in crude membrane fractions of Neurospora crassa mycelia. Here we report the isolation and characterization of a mutant (psp; phosphorylation of small proteins that is completely defective for phosphorylation of that protein, as assayed in both crude membrane and soluble fractions. This mutation defines a unique locus that maps to linkage group VR between al-3 and his-6. To elucidate the photobiological significance of the phosphorylation of the protein, we analyzed known photobiological phenomena and discovered that the positioning of beaks on the perithecia, defined as perithecial polarity, was light-dependent in the wild type. In the psp mutant, beaks were phototropic as in the wild type, but their position was random. In a wc-l mutant, however, beaks were positioned at random and were not phototropic. Thus light-induced perithecial polarity and phototropism of perithecial beaks are controlled differently. A psp; wc-l double mutant showed the same phenotype as that of wc-l with respect to these two photomorphogenetic characters. These results indicate that the wc-l gene is epistatic to psp in the light-signal transduction pathway that controls both phototropism and perithecial polarity.     

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Tissue-specific repression of starvation and stress responses of the Neurospora crassa con-10 gene is mediated by RCO1

Abstract-This study presents an evaluation of the effectiveness of the AIDS Community-Based Outreach/Intervention projects implemented as part of the National Institute on Drug Abuse (NIDA) Cooperative Agreement (CA), which began in 1990 and is currently ongoing. Participants in the CA were randomly assigned to one of two interventions: a NIDA/CA-developed standard intervention (SI); or the SI plus a site-specific enhanced intervention (EI). Analyses of drug use and needle-related risk behaviors were conducted among injection drug users (IDUs) in eight participating cities where follow-up rates of at least 60% were obtained (N=3,743). Results indicated that IDUs significantly reduced their needle-related risk behaviors following delivery of the interventions and that a substantial portion entered substance abuse treatment. However, there was relatively little to support the effectiveness of more expensive and involved enhanced interventions. A number of factors associated with increasing or maintaining high risk behaviors, including an HIV negative serostatus and a greater perceived chance of acquiring AIDS, were also observed. Continued outreach to drug injectors is recommended, as well as the development of new and creative interventions targeting individuals who are HIV negative and those who are aware of their high risk status but have not changed their behaviors in response to risk-reduction interventions.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.     

Analysis of electrical phenomena accompanying the growth of Neurospora crassa hyphae: theory and experiment

To describe electrical phenomena observed in growth of Neurospora crassa hyphae, a theoretical model was developed considering the hypha as a one-dimensional electric cable with non-uniform longitudinal distribution of current sources reflecting the activity of proton pumps. A profile of the density of the pump current along the hypha is proposed, at which the results of simulation quantitatively coincide with the results of physiological experiments. The model values of energy coupling in the growth zones were estimated. The experimental dependence of the elongation rate of regenerating apical hypha fragments on their lengths was determined. Based on the comparison of these experimental results with the results of analysis of the model, the contribution of the axial metabolite transport, from the distal parts of the hypha to the apical part, to the dynamics of the apical cell growth was estimated. The possibility of evaluating the intensity of high-molecular-weight syntheses and/or accumulation of substances in granules was demonstrated. The growth rate of the regenerating hypha fragments was shown to correlate with the electric current flowing into the apical fragment 0.2-mm in length.     

rco-3, a gene involved in glucose transport and conidiation in Neurospora crassa

Macroconidiation in Neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. Conidia formation is normally observed when the fungus is exposed to air. However, carbon limitation can induce conidiation in mycclia submerged in an aerated liquid medium. A mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been cloned. RCO3 exhibits sequence similarity to members of the sugar transporter gene superfamily, with greatest similarity to glucose transporters of yeast. Consistent with this structural similarity, we find that glucose transport activity is altered in the mutant. However, growth of the mutant in media containing alternate carbon sources does not suppress conidiation in submerged culture. The properties of the mutant suggest that RCO3 is required for expression of glucose transport activity, glucose regulation of gene expression, and general carbon repression of development.     

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.     

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa

Lattice-like perineuronal accumulations of extracellular-matrix proteoglycans have been shown to develop during postnatal maturation and to persist throughout life as perineuronal nets (PNs) in many brain regions. However, the dynamics of their reorganization in adults are as yet unknown. The aim of the present study was to examine the capability of PNs for reconstitution after experimental destruction and to search for possible consequences of extracellular-matrix degradation for neurons and glial cells. The changes were induced by single intracortical injections of Proteus vulgaris chondroitinase ABC and studied after postinjection periods of 1 day to 5 months. The N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA), an antibody against chondroitin-sulphate proteoglycans, three antibodies recognizing initial chondroitin or chondroitin-sulphate moieties ('stubs') of proteoglycan core proteins, an antibody against the hyaluronan-binding protein component of versican, and biotinylated hyaluronectin, which binds to hyaluronan, were used as cytochemical markers. One day postinjection, the WFA-binding sites and hyaluronan were shown to be almost completely removed within a circumscribed digestion zone. The staining of different core-protein components revealed only fragments of PNs. These changes were found to be partly compensated 4 weeks after injection of chondroitinase ABC. After 8 and 12 weeks postinjection, the cytochemical and structural characteristics as well as the area-specific distribution patterns of PNs were progressively reconstituted. At 5 months postinjection, they could not be distinguished from those in untreated tissue. In contrast to such transient changes, a diffuse chondroitin-sulphate proteoglycan immunoreactivity persisted in the neuropil. Loss of neurons or alterations of their structure as well as reactions of glial cells were not observed. We conclude from this study that PNs, enzymatically destroyed in the adult rat brain, can be completely reconstituted, but the restoration of their extracellular-matrix components needs several months.