A comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type Neurospora crassa strain and mutants, white collar-1 and white collar-2

A comparative study of the changes in the components of the antioxidant defense system (ADS), the activity of superoxide dismutase (SOD) and catalase and the level of extractable SH-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) Neurospora crassa strains was performed. Oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable SH-groups and SOD activity, whereas the total catalase activity decreased during growth. However, in contrast to the wild-type strain, the activity of the catalase in the mutant strains wc-1 and wc-2 slightly increased upon the transition to the stationary phase. In the wc-2 mutant, SOD activity and the level of extractable SH-groups in the exponential growth phase were always lower than in the wild-type and wc-2 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.     

Stability of chitin synthetase in cell-free preparations of a wild-type strain and a ''slime'' variant of Neurospora crassa

Chitin synthetase activity in cell-free preparations from a wild-type strain and a 'slime' variant of Neurospora crassa was monitored over many days in samples stored at 0 degrees C. Total activity in whole-cell-free extracts and low-speed supernatants from both organisms was very unstable, losing more than 90% of the initial activity on storage at 0 degrees C for 96 h. Chitin synthetase detection was not masked by chitinase activity present in the preparations. Gel-filtration chromatography of these preparations increased the stability of the activity from the 'slime' variant, whereas removal of particulate structures by high-speed centrifugation stabilized the chitin synthetase activity in the supernatant, particularly in the wild type. These results suggest that factor(s) involved in the regulation of chitin synthetase may be differentially located or altered in 'slime' cells.     

Copper accumulation in the cell-wall-deficient slime variant of Neurospora crassa. Comparison with a wild-type strain.

The copper-uptake process in the cell-wall-deficient slime variant of the fungus Neurospora crassa was compared with that in a wild-type strain. In both organisms investigated most of the copper is taken up from the culture medium during the exponential growth period. The wild-type strain, however, accumulates much more copper than does the slime variant. The influence of the copper concentration in the culture medium on the amounts of copper accumulated intracellularly suggests separate ways of copper import used by the two morphologically different N. crassa strains. Copper analyses of three different cytosolic fractions as a function of growth time or exogenous copper concentration indicate both strains to share a very similar copper metabolism. All the data presented are consistent with a detoxification function of the low-Mr copper-binding fraction of N. crassa. Both copper-metallothionein and oxidized glutathione (GSSG) are co-eluted with this fraction. The possible involvement of glutathione in metallothionein biosynthesis is discussed.     

A comparative study of DNA-induced transformants and spontaneous revertants of inositolless Neurospora crassa.

Inositolless (inl-) Neurospora crassa strains were treated with DNA (allo-DNA) of wild type N. Crassa. Hyphal fragments of a mycelial suspension of the N. Crassa ragged inl- strain used as recipient in our transformation experiments were found to consist of units containing 100--1000 nuclei. In this strain the inositol-independent (inl+) nuclei appearing after DNA treatment or by spontaneous reversion are present in the cytoplasm together with a large number of inl- nuclei. Thus, both transformation and reversion initially must result in heterokaryosis. Under appropriate conditions the inl- nuclei can be detected in the transformed and spontaneous inl+ phenotype revertant strains. Spontaneous revertants are usually characterized by the loss of their inl+ nuclei after transfers on inositol-supplemented medium. On minimal medium, the growth rate of transformed strains is significantly lower than that of spontaneous revertants. The inl+ gene appearing after DNA treatment or by spontaneous reversion is inherited as a trait bound to chromosomes. In crosses with the transformed strains, there is a significant increase in the number of non-Mendelian (6:2 and 5:3) tetrads in the inl locus.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of Neurospora crassa and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-1 (wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction in the WCC mutants of stress factor signals that promote ROS (reactive oxygen species) formation.     

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

Summary A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35°C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40°C indicated that the maximal growth rate occurs at 35°C in the wild type, and at 25°C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35°C than at 25°C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20°C is not sufficient to allow germination at 37°C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37°C. The reciprocal temperature shift-down experiments showed that the molecular events at 37°C may be qualitatively useful for germination after shifting to 20°C. Studies of macromolecular synthesis showed that the biochemical defect in the heat-sensitive strain appears to affect RNA synthesis before protein synthesis, although there were differences in the relative effects depending on the age of the germinating conidia and the inhibition of the two processes was never complete. Messenger RNA synthesis is normal in the mutant at 37°C. Previous work has shown that the rip-1 mutant strain has a conditional defect in the accumulation of 25S rRNA and, hence, in 60S ribosomal subunit production (Loo et al. 1981). There are also indications from those studies that the 60S ribosomal subunit may be functionally impaired at the higher temperature. Thus, the growth and macromolecular synthesis phenotypes may result as a consequence of a conditional, ribosome function defect and leads to the hypothesis that the mutation in the rip-1 strain may be in a gene for a 60S ribosomal subunit component, perhaps a ribosomal protein.     

Sterol mutants of Neurospora crassa: their isolation, growth characteristics and resistance to polyene antibiotics

Summary A method is described for isolating sterol mutants of the filamentous fungus, Neurospora crassa. Most of the mutants carry gene mutations affecting the later stages of ergosterol biosynthesis and they accumulate other, as yet unidentified, sterol components but two mutants are blocked earlier in the pathway and respond to exogenous mevalonic acid. Altered sterol metabolism is associated with a reduced rate of growth, abnormal morphology, poor fertility and resistance to a variety of polyene antibiotics.     

Uptake, intracellular binding, and excretion of polyamines during growth of Neurospora crassa

In Neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. We wished to know what determines these pool sizes. In the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. In a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. However, this was true only after an intracellular "sink," with a capacity equal to the amount of spermidine found in wild-type cells, had been saturated. We speculate that internal anionic binding sites, detectable in permeabilized cells, sequester virtually all of the spermidine normally found in exponentially growing N. crassa. Further evidence for this view was that in mature, stationary cultures, excess spermidine is excreted. Putrescine is also excreted if its concentration in the cell is abnormally high. The control of pool size by intracellular binding and excretion may be an advantage in this pathway, because feedback inhibition does not prevail, enzyme regulation is by comparison slow, and excessive polyamines are toxic.     

Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques.

Genetic and complementation mapping studies using 20 qa-2 mutants defective for catabolic dehydroquinase indicate that the qa-2 gene encodes a single polypeptide chain and is the structural gene for catabolic dehydroquinase, a 220,000-molecular-weight protein composed of identical 10,000-molecular-weight subunits. Many qa-2 mutants are capable of reversion, but no evidence has yet been obtained for nonsense mutations in this gene. The biochemical consequences of the mutations in two complementing qa-2 strains (M239 and M204) have been determined. Both mutants have extremely low levels of catalytic activity and form a heterocaryon with about 4% of the wild-type activity. As assayed by immunological cross-reactivity, mutant M239 and the heterocaryon have nearly wild-type levels of native-molecular-weight catabolic dehydroquinase protein, whereas M204 has no detectable amount of this protein. Thus it is concluded that M239 has a mutation at or near the catalytic site which reduces the activity 10,000-fold but has little or no influence on the formation of the native multimeric structure. In contrast, M204 apparently has a mutation that severely inhibits aggregation and may have only a minor effect on the inherent potential for catalytic conversion at the reactive site. The heterocaryon would appear to form a mixed multimer with the monomeric subunits from M239 providing the aggregated structure and those from M204, the catalytically active moiety.     

一株粗糙脉孢菌的分离、鉴定及其基因组变异和功能分析

【背景】粗糙脉孢菌LY03是从武平传统"红菌豆腐"中分离得到的主要发酵菌株。【目的】研究粗糙脉孢菌LY03菌株的基因组信息,揭示武平传统"红菌豆腐"发酵特性。【方法】采用形态学观察、ITS鉴定、重测序及框架图测序对所分离的LY03菌株进行鉴定和基因组信息解析。【结果】武平传统"红菌豆腐"中分离得到的主要发酵菌株LY03确定为粗糙脉孢菌,将其在中国普通微生物菌种保藏管理中心进行专利保藏,保藏号为CGMCC3.19233。LY03菌株比对到参考基因组上的总Read数目为95.85%,测序对应深度的位点占全基因组76.13%;各变异类型在内含子区域均无变异,主要变异存在于基因组的外显子区域,具体变异数量为:单核苷酸多态性位点(single nucleotide polymorphism,SNP)位点变异总数203128个、插入缺失(insertion/deletion,In Del)突变总和26859个、拷贝数变异(copy number variation,CNV)增加和减少的拷贝总数1 039个、结构变异(structure variation,SV)注释的变异总数777个;LY03菌株...     

Growth,respiratory, and cytochrome characteristics of certain of the isoleucine-valine mutants of Neurospora crassa

Several of the isoleucine-valine requiring mutants of Neurospora crassa have been shown to have differences in growth rate, oxygen uptake rate, cyanide sensitivity, and cytochrome spectral patterns as compared to wild type. This pleiotropic effect seems to be true for mutants with changes at all three known loci affecting isoleucine and valine biosynthesis, the iv-1, iv-2, and iv-3 loci.     

Molecular and kinetic study of catalase-1, a durable large catalase of Neurospora crassa.

Catalase-1 (Cat-1), one of the two monofunctional catalases of Neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. Cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. Like most monofunctional catalases, Cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a pH range of 4-12, and inactivated by 3-amino-1,2,4-triazole. Unlike most monofunctional catalases, Cat-1 consists of 88 kDa monomers that are glycosylated with alpha-glucose and/or alpha-mannose, is unusually stable, and is not inactivated or inhibited by hydrogen peroxide. Cat-1 was more resistant than other catalases to heat inactivation and to high concentrations of salt and denaturants. Cat-1 exhibited unusual kinetics: at molar concentrations of hydrogen peroxide the apparent V was 10 times higher than at millimolar concentrations. Inactivation of Cat-1 activity with azide and hydroxylamine was according to first order kinetics, while cyanide at micromolar concentrations was a reversible competitive inhibitor.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Epistasis, photoreactivation and mutagen sensitivity of DNA repair mutants upr-1 and mus-26 in Neurospora crassa

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.