Translesion DNA polymerases Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa.

Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol zeta, is a highly polymorphic locus in Neurospora.

Identification of non-specific alkaline phosphatases in hyphal cells of the fungus Neurospora crassa by in situ histochemistry

The present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus Neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. Our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either Mg2+ or EDTA. Another alkaline phosphatase activity, whose location in the hyphal cell is regulated by phosphate, is stimulated by Mg2+, inhibited by EDTA, and somehow dependent on the expression of the pho-2+-encoded Pi-repressible alkaline phosphatase.

Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon of Neurospora crassa.

Heterokaryons of Neurospora crassa were generated by transformation of multinucleate conidia of a histidine-3 auxotroph with his-3(+) plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained an ectopically integrated his-3(+) allele. This response was specific to histidine. The reduction in prototrophic nuclei was confirmed by several criteria: inoculum size test, hyphal tip analysis, genomic Southern analysis, and by visual change in colour of the transformant incorporating genetic colour markers. Construction and analyses of three-component heterokaryons revealed that the change in nuclear ratio resulted from interaction of auxotrophic nucleus with prototrophic nucleus that contained an ectopically integrated his-3(+) gene, but not with prototrophic nucleus that contained his-3(+) gene at the normal chromosomal location. The growth rate of heterokaryons and the activity of histidinol dehydrogenase - the protein encoded by the his-3(+) gene - remained unchanged despite prototrophic nuclei becoming very scarce. The results suggest that not all nuclei in the coenocytic fungal mycelium may be active simultaneously, the rare active nuclei being sufficient to confer the wild-type phenotype.

Genes encoding chimeras of Neurospora crassa erg-3 and human TM7SF2 proteins fail to complement Neurospora and yeast sterol C-14 reductase mutants.

The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this enzyme is encoded by the erg-3 and erg24 genes respectively. In an effort to demonstrate sterol C-14 reductase activity for SR-1 we constructed six recombinant genes coding for chimeras of the Neurospora erg-3 and SR-1 protein sequences and tested them for complementation of the Neurospora erg-3 mutant. To our surprise, all the chimeras failed to complement erg-3. A few of the chimeric proteins were also tested against the yeast erg24 mutant, but again there was no complementation. We discuss some reasons that might account for these unexpected findings.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism

Purification and characterization of flavokinase from Neurospora crassa.

The ATP-dependent phosphorylation of riboflavin to FMN by flavokinase is the key step in flavin biosynthesis. Flavokinase has been purified from a fungal source for the first time. The enzyme purified from a cell wall lacking mutant of Neurospora crassa, slime, is a monomer of M(r) 35.5 kDa with maximal activity at alkaline pH and high temperature (55 degrees C). The K(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nM for riboflavin and 210 nM for MgATP2-). The enzyme exhibits preference for Mg2+ over Zn2+ as the essential activator and is also significantly activated by several cations. Activation by orthophosphate may be physiologically relevant for the intracellular regulation of flavokinase.

Serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from Neurospora crassa

The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type 1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PPI activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor- 1 (lC50 2 nM). On the other hand, PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-l. About 80 per cent of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.

A hyperoxidant state at the start of each developmental stage during Neurospora crassa conidiation

Neurospora crassa conidiation and other microorganism differentiation processes can be explained as a response to a hyperoxidant state. Many proteins, among them NADP-glutamate dehydrogenase and glutamine synthetase, were oxidatively modiried and degraded at the start of all three morphogenetic transitions of the conidiation process. A shift in the redox state of the main cellular electron carriers, from a highly reduced state to a predominantly oxidized one, occurred stepwise with a highly oxidizing event at the start of each transition. Catalase activity increased during cell differentiation in various microorganisms and one of two catalases accumulated to a high level in the differentiated cell. Catalases in Neurospora were oxidized in vitro and in vivo giving rise to active enzyme conformers. Carotenoids, which have antioxidant functions in fungi, were consumed in each morphogenetic stage and oxidized carotenoids accumulated in differentiated cells. Carotenoid biosynthesis was induced following oxidative stress and during conidiation in each new cell structure. Increased generation of reactive oxygen species was detected by chemiluminescence at the onset of all transitions during differentiation. Antioxidants inhibited both light emission and cell differentiation. These and other data from the literature are discussed in the context of the Dioxygen Avoidance Theory of cell differentiation.

Ingeniería genética en hongos filamentosos: clonamiento del gen de invertasa de neurospora crassa / Genetic engineering in filamentous fungi: cloning of the invertase gene from Neurospora crassa

The invertase wild type gene of N. crassa was cloned into the YRp7 yeast vector. This recombinant plasmid was selected by functional complementation of an invertaseless mutant strain of S. cerevisiae. The isolated recombinant plasmid (named pNC2) carries a 7.6 Kb BamHI DNA fragment from N. crassa. The cloned DNA hydbridized with the N. crassa genomic DNA and transformed an invertase mutant of N. crassa Inv- to Inv+. Transformation of N. crassa Inv- to Inv+ seems to take at least two different integration events. One of them involves an integration closely linked to inv locus, and the other one apparently involves as integration of cloned DNA at a genomic site different that the inv locus

Liberaçäo das fosfatases ácida e alcalina repressíveis de Neurospora grassa por detergentes e sais / Liberation of repressibles acid and alkaline phosphatases from Neurospora grassa by detergents and salts

O efeito de diversos compostos químicos sobre a liberaçäo das fosatases ácidas e alcalina repressíveis de Neurospora crassa foi estudado. A fosfatase ácida teve sua atividade estimulada quando adicionaram ao meio de cultura Twee 80, Tween 20 e dodecilsulfato de sódio (SDS) enquanto que a atividade da fosfatase alcalina foi favorecida por Twen 80, Tween 20, SDS, desoxicolato de sódio, NaCl e KCl. Além do efeito sobre a liberaçäo das enzimas, os detergentes näo iônicos, Tween 80, Tween 20 e Triton X-100, adicionados à mistura de reaçäo, estimularam a atividade da fosfatase ácida enquanto que os iônicos, taurocolato de sódio e SDS, a inibiram. Esse efeito näo foi observado com a fosfatase alcalina. Concentraçöes crescentes de Tween 80 aumentaram a secreçäo da fosfatase ácida e de proteína. A liberaçäo da fosfatase ácida foi dependente do tempo, culminando a atividade da enzima após 3 horas de adiçäo do Tween 80

Acid phosphatase (EC 3.1.3.2) synthesis by phosphorus regulatory mutant strains of Neurospora crassa

1. Even though altered forms of acid phosphatase II were wynthesixed by the mutant strains nuc-1A and nuc-2A of N. crassa, their synthesis was independent of exogenous phosphate concentratiosn. 2. Synthesis of acid phosphatase I by nuc-2A was also insensitive to exogenous phosphate concentrations. When nuc-1A was grown on a low-phosphate medium, it also produced a heat-labile acid phosphatase in addition to a I-like acid phosphatase. I-like acid phosphatase was not detected in the mycelium of the preg*c mutant strain grown on low-phosphate medium. 3. These results are consitent with participation of the nuc-2, preg and nuc-1 genes in regulating the transport and/or secretion of acid phosphatase and probably other phosphatases by Neurospora crassa