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Four new illudane derivatives with antibiotic and cytotoxic properties, illudins I (1), I(2) (2), J (3) and J(2) (4), have been isolated from the fungus Coprinopsis episcopalis (syn. Coprinus episcopalis). These sesquiterpenes are stereoisomers, and their relative structures have been determined taking into consideration 2D NMR data.
One of the most basic functions in any organism is DNA repair. In addition, programmed DNA "damage," in the form of DNA double-strand breaks (DSBs), is a regular part of the physiology of most organisms. There are three main types of DSB repair: homologous recombination; single-strand annealing; and nonhomologous end joining. The gene products known to be required for these repair processes are conserved in evolution, but the relative dependence on different pathways for DSB repair is different when systems are compared. In the yeast Saccharomyces cerevisiae, the formation and repair of DNA double-strand breaks (DSBs) is apparently an essential feature of meiotic recombination. However, it is not clear whether DSBs are a conserved feature of meiotic recombination in eukaryotes. The basidiomycete Coprinus cinereus presents an experimental system which is amenable to genetic analysis, processes DSBs in a manner similar to complex eukaryotes, and has a naturally synchronous meiosis. An understanding of the functions of conserved genes in DSB repair in C. cinereus and other similar systems will help to determine whether DSB repair is a unifying theme in meiotic recombination or whether conserved gene products have other essential functions that tie together DNA repair and meiosis.
Mathematical models which imitate plant gravitropic responses were used to compare plant and fungal gravitropism with kinetic data from the agarics Coprinus cinereus and Flammulina velutipes. Similarities were: bending depends on differential growth; growth of the organ is most intensive just behind the apex; gravitropisms exhibit a substantial time delay. Differences were: the agaric stem apex always returns to the vertical (some plant organs show stable plagiogravitropic growth); curvature compensation occurred in C. cinereus; C. cinereus stems rarely overshot or oscillated around the vertical although data for F. velutipes showed a single overshoot and oscillation. The work focused attention on the need for data on detection-level thresholds, angle-response and acceleration-response relationships in fungi, and the need for detailed observations of gravitropism kinetics in a larger number and wider range of fungi.
Several fungi were screened for protein production in a simple medium using either sugarcane bagasse, wheat straw or cowdung as substrate. Out of them Rhizoctonia melongina, Pleurotus ostreatus, and Coprinus aratus proved promising and were taken up for further studies. Optimum conditions of temperature, incubation, and pH for maximum growth and protein production by the three fungi were then ascertained and standardized.
An antifungal protein, with a molecular weight of 15 kDa and an N-terminal sequence analogous to those of chitinases, was first isolated from the Chinese medicinal material Panax notoginseng, using cation exchange chromatography and affinity chromatography. The protein was adsorbed on CM-cellulose, Affi-gel Blue Gel and Mono S. It exerted antifungal activity against Coprinus comatus, Fusarium oxysporum and Mycosphaerella arachidicola but not against Rhizoctonia solani. The protein was devoid of ribonuclease activity against yeast tRNA.
We hybridized the telomere-associated DNA sequence pTel46 isolated from Coprinus cinereus with Helicobasidium mompa genomic DNA. The hybridized fragments were more sensitive to Bal31 nuclease than those that were not hybridized, suggesting that they were located at the ends of chromosomes in H. mompa. The hybridization profile can be used to estimate chromosome number, since the number of chromosomes in a single basidiospore isolate is about half that in putative parent strains. Thus, single basidiospore and field isolates might be homokaryons and heterokaryons respectively. We found telomere-linked restriction fragment length polymorphisms (RFLPs) in strains of H. mompa isolated from field and individual basidiosporcs. Thus, this marker appears to be an excellent tool with which to reveal the considerable polymorphism of H. mompa and to identify strains. The RFLP was not found among several strains of the same mycelial compatibility group (MCG) isolated from the same field, suggesting that strains belonging to an MCG group are identical.
Podospora anserina and Coprinopsis cinerea (syn. Coprinus cinereus) are endowed with a defence system able to differentiate self vs. non-self and involving the generation of peroxide. Indeed, they produce peroxide when confronted with a filamentous fungus, only in non-self confrontations. Both species are not able to recognize yeasts and show a differential response to bacteria. The accumulation of peroxides in the ascomycete Podospora anserina requires an NADPH oxidase and a MAP kinase cascade, previously shown to be involved in fruit body formation, cell differentiation and cell degeneration. Confrontation is accompanied by the death of the contestant hyphae only in specific combinations of species. As in animals and plants, data suggest that peroxide is likely involved in signalling rather than playing a direct toxic role. Fungi display more complex behaviours than generally acknowledged, i.e. they are able to recognize potential contestants and built up defence reactions involving evolutionary conserved enzymes.
The peroxidase-catalyzed decomposition of 3-hydroperoxy-1-butene (1), 2,3-dimethyl-3-hydroperoxy-1-butene (2), tert-butyl hydroperoxide (3), ethyl oleate hydroperoxide 4, and linoleic acid hydroperoxide 5 was applied as a chemical model system to assess whether lipid hydroperoxides may cause DNA damage under peroxidase catalysis. For this purpose, the Coprinus peroxidase (CIP), horseradish peroxidase (HRP), and the physiologically important lactoperoxidase (LP) were tested. Indeed, hydroperoxides 1-5 induce strand breaks in pBR 322 DNA upon peroxidase catalysis. For the nucleoside dG, the enzymatic decomposition of hydroperoxides 1-4 led to significant amounts of 4, 8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (4-HO-8-oxo-dG) and guanidine-releasing products (GRP), whereas 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was not obtained. In isolated calf thymus DNA, the efficient conversion of the guanine base (Gua) was observed. Peroxyl radicals, which are generated in situ from the hydroperoxides by one-electron oxidation with the peroxidases, are proposed as the active oxidants on the basis of the following experimental facts. (i) Radical scavengers strongly inhibit the guanine oxidation in dG and DNA and strand-break formation in the latter. (ii) EPR spectral studies with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap confirmed the formation of peroxyl radicals. (iii) The release of molecular oxygen was demonstrated, produced through the disproportionation of peroxyl radicals. The biological relevance of these findings should be seen in the potential role of the combined action of lipid hydroperoxides and peroxidases in damaging cellular DNA through peroxyl radicals.
A homodimeric lectin adsorbed on Affi-gel blue gel and CM-Sepharose and possessing a molecular weight of 67 kDa was isolated from red kidney beans. The hemagglutinating activity of this lectin was inhibited by glycoproteins but not by simple sugars. The lectin manifested inhibitory activity on human immunodeficiency virus-1 reverse transcriptase and alpha-glucosidase. The N-terminal sequence of the lectin exhibited some differences from previously reported lectins from Phaseolus vulgaris but showed some similarity to chitinases. It exerted a suppressive effect on growth of the fungal species Fusarium oxysporum, Coprinus comatus, and Rhizoctonia solani. The lectin had low ribonuclease and negligible translation-inhibitory activities.
The kinetics of Coprinus cinereus peroxidase-catalyzed 1-naphthol, 2-naphthol, and 4-hydroxybiphenyl oxidation was investigated. The initial rates of the naphthols' and 4-hydroxybiphenyl oxidations were linearly dependent on enzyme concentration. The rates depended on substrate concentration and saturated at concentrations above 100 microM of hydrogen peroxide, 25-50 microM of naphthols, and 10 microM of 4-hydroxybiphenyl. At the peroxide concentration 100 microM calculated K(m) and the maximal rate (V(max)) were 74.7 microM and 0.53 microM/sec or 175 microM and 2.0 microM/sec for 1- or 2-naphthol, respectively, and 29.68 microM and 0.42 microM/sec for 4-hydroxybiphenyl. Kinetic measurements of exhaustive naphthol and 4-hydroxybiphenyl oxidation showed that peroxidase is inactivated during the oxidation of the substrates. Different factors and additives, water soluble polymers and albumins (PEG, PEI, PL, BSA, HSA), influenced the initial naphthols and 4-hydroxybiphenyl oxidation rates, peroxidase inactivation rates, and the degree of the substrate conversion. Addition of albumin increased turnover number of naphthols oxidation 1.5-4 times. Light scattering increase was observed when peroxidase-catalyzed oxidation reaction was investigated and suggested that insoluble particles were formed during the process. The addition of polymers, change of concentration and ionic strength of the solution as well as the number of other factors influenced the observed light scattering. The number of particles formed during peroxidase-catalyzed naphthols' and 4-hydroxybiphenyl oxidation and their distribution according to size in the interval 2.5-300 microm were detected by particle counting in solutions.
The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.
We have crystallized the ascomycete laccase from Melanocarpus albomyces with all four coppers present and determined the crystal structure at 2.4 A resolution. The enzyme is heavily glycosylated and consists of three cupredoxin-like domains, similar to those found in the Cu-depleted basidiomycete laccase from Coprinus cinereus. However, there are significant differences in the loops forming the substrate-binding pocket. In addition, the crystal structure of the M. albomyces laccase revealed elongated electron density between all three coppers in the trinuclear copper site, suggesting that an oxygen molecule binds with a novel geometry. This oxygen, required in the reaction, may enter the trinuclear site through the tunnel, which is open in the structure of the C. cinereus laccase. In contrast, the C-terminus on the M. albomyces laccase forms a plug that blocks this access.
The assumption that the mushroom stem has the ability to undergo autonomic straightening enables a mathematical model to be written that accurately mimics the gravitropic reaction of the stems of Coprinus cinereus. The straightening mechanism is called curvature compensation here, but is equivalent to the 'autotropism' that often accompanies the gravitropic reactions of axial organs in plants. In the consequently revised local curvature distribution model, local bending rate is determined by the difference between the 'bending signal' (generated by gravitropic signal perception systems) and the 'straightening signal' (proportional to the local curvature at the given point). The model describes gravitropic stem bending in the standard assay with great accuracy but has the virtue of operating well outside the experimental data set used in its derivation. It is shown, for example, that the mathematical model can be fitted to the gravitropic reactions of stems treated with metabolic inhibitors by a change of parameters that parallel the independently derived physiological interpretation of inhibitor action. The revised local curvature distribution model promises to be a predictive tool in the further analysis of gravitropism in mushrooms.
The roots of the sanchi ginseng, Panax notoginseng, were extracted with an aqueous buffer. The extract was chromatographed on a CM-cellulose column to remove extraneous unadsorbed proteins. The adsorbed fraction was dialyzed and chromatographed on Affi-gel blue gel. The adsorbed fraction was again collected, dialyzed and applied on a column of Mono S. The second peak was dialyzed and chromatographed on an FPLC-gel filtration Superdex 75 column. An antifungal protein with an N-terminal sequence similar to those of chitinases was isolated from the first peak which had a molecular mass of 35 kDa. The sequence was distinctive in that the third and ninth highly conserved N-terminal residues (C and G) were replaced by H and M, respectively. The protein inhibited mycelial growth in Coprinus comatus, Physalospora piricola, Botrytis cinerea, and Fusarium oxysporum with an IC 50 of 100 nM, 1 microM, 630 nM and 560 nM, respectively. It inhibited cell-free translation with an IC 50 of 630 nM. Its antifungal and translation-inhibitory activities were more potent than those of previously reported antifungal proteins. It inhibited human immunodeficiency virus-1 reverse transcriptase by 35.8 % at 12.6 microM and 24.7 % at 1.26 microM.
Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.
A key problem in evolutionary biology has been distinguishing the contributions of current and historical processes to the maintenance of genetic variation. Because alleles at self-recognition genes are under balancing selection, they exhibit extended residence times in populations and thus may provide unique insight into population demographic history. However, evidence for balancing selection and extended residence times has almost exclusively depended on identification of transspecific polymorphisms; polymorphisms retained in populations through speciation events. We present a broadly applicable approach for detecting balancing selection and apply it to the b1 mating type gene in the mushroom fungus Coprinus cinereus. The comparison of neutral molecular variation within and between allelic classes was used to directly estimate the strength of balancing selection. Different allelic classes are defined as encoding different mating compatibility types and are thus potentially subject to balancing selection. Variation within an allelic class, where all alleles have the same mating compatibility type, provided an internal standard of neutral evolution. Mating compatibility in this organism is determined by the complex A mating type locus, and b1 is one of several redundantly functioning genes. Consequently, we conducted numerical simulations of a model with two subloci and varying levels of recombination to show that balancing selection should operate at each sublocus. Empirical data show that strong balancing selection has indeed occurred at the b1 locus. The widespread geographic distribution of identical b1 alleles suggests that their association with differing A mating types is the result of recent recombination events.
Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.
Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein-protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.