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PubMed1993

A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization.

A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)12-18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.

Aphidicolin↗
PubMed1983

Structure of a ribosomal 5S RNA from a mushroom, Coprinus cinereus.

The nucleotide sequence of the 5S rRNA from a mushroom, Coprinus cinereus, was determined to be: (sequence in text). This sequence is consistent with current models for the secondary structure of 5S RNAs and indicates a very high degree of sequence conservation among the most highly evolved fungi. Sequence heterogeneity was not evident in this fungus suggesting that the more highly evolved fungi may not contain the dispersed pattern of 5S rRNA genes which have been observed in intermediate fungi such as Neurospora.

Base Sequence↗
PubMed1992

Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding gene from the basidiomycete Coprinus cinereus.

The naturally synchronous meiosis of the fungus, Coprinus cinereus, provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a lambda gt11 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene (MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.

Amino Acid Sequence↗
PubMed2003

Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi.

The illudins are a family of fungal sesquiterpenes that have been studied as anti-tumor agents, and they also have antibacterial activity. Over a four-year period, 25 304 fungal isolates (approximately 97% ascomycetes and 3% basidiomycetes), were screened for antibacterial activity against methicillin-resistant Staphylococcus aureus. Illudin-like compounds with antibacterial and cytotoxic activity against tumor cell lines were observed in 10 basidiomycete strains. The isolates were recovered from different types of substrata using indirect methods and only formed sterile mycelia in pure culture. The isolates were genetically related but not identical, based on PCR-based fingerprinting techniques. DNA sequencing of the ITS1-5.8 S-ITS2 region of the strains revealed that nine had identical sequences, indicating that they were conspecific. The sequence of the remaining isolate was 96.34% similar, suggesting that it was a closely related species. The D1-D2 region of the 25 S rRNA gene of the two strain types was also sequenced. Both sequences were 99.39% similar, and Coprinopsis gonophylla (syn. Coprinus gonophyllus) was the closest match for both. Strains were grown in pure culture on a rice-based medium that allowed the development of basidiomata from one culture of the main strain type, which was identified as C. episcopalis, a close relative of C. gonophyllus. Both species (or strain types) produced different types of illudin-like compounds. Three novel illudins (I, I2 and J2) were found to be produced by the cultures identified as C. episcopalis, while only illudinic acid was produced by the other Coprinopsis sp. The taxonomical relationships of the Coprinops is species identified in this study with other illudin producers previously reported in the literature are discussed.

Agaricales↗
PubMed1998

Turnover capacity of coprinus cinereus peroxidase for phenol and monosubstituted phenols

Coprinus cinereus peroxidase (CIP) and other peroxidases are susceptible to mechanism-based inactivation during the oxidation of phenolic substrates. The turnover capacity (defined as the molar or mass concentration of substrate oxidized per unit concentration of enzyme inactivated) of CIP was quantified for phenol and 11 monosubstituted phenols under conditions in which enzyme inactivation by mechanisms involving hydrogen peroxide alone were minimized. Turnover capacities varied by nearly 2 orders of magnitude (absolute values on the order of 10(5)-10(6) on a molar basis), depending on the substituent. On a mass basis, the enzyme consumption corresponding to the lowest turnover capacities is considerable and may influence the economic feasibility of proposed industrial applications of peroxidases. Within a range of substituent electronegativity values, molar turnover capacities correlated well (r2 = 0.89) with substituent effects quantified by radical sigma values and semiquantitatively with homolytic O-H bond dissociation energies of the phenolic substrates, suggesting that phenoxyl radical intermediates are probably involved in the suicide inactivation of CIP. The correlation range in each case did not include phenols with highly electron-withdrawing (nitro and cyano) substituents because they are not oxidized by CIP, nor phenols with highly electron-donating (hydroxy and amino) substituents because they led to virtually complete inactivation of the enzyme with minimal substrate removal. In the latter case we conclude that inactivation of CIP during the oxidation of hydroxy- and amino-substituted phenols occurs by a different mechanism than that of the other phenolic substrates.

Journal Article↗
PubMed1999

Asymmetric Synthesis with the Enzyme Coprinus Peroxidase: Kinetic Resolution of Chiral Hydroperoxides and Enantioselective Sulfoxidation.

The enzyme Coprinus peroxidase (CiP) was employed for the kinetic resolution of racemic hydroperoxides 1 and the asymmetric sulfoxidation of prochiral sulfides 4. Eleven hydroperoxides 1a-k were reduced by CiP and guaiacol as reductant under conditions of kinetic resolution with enantioselectivities of up to >98% for the (S)-hydroperoxide 1 and 90% for the (R)-alcohol 2. In the absence of a reductant, the hydroperoxide 1a afforded with CiP enantiomerically enriched hydroperoxide 1a (ee up to 54%) and alcohol 2a (ee up to 40%), as well as ketone 3a (which is also formed simultaneously in all other reactions) and molecular oxygen. Catalase activity was established for CiP with hydrogen peroxide. When aryl alkyl sulfides 4 were used as oxygen acceptors, three products, sulfoxides 5, alcohols 2, and hydroperoxides 1, were obtained, all in enantiomerically enriched form. The highest ee value (89%) was achieved for the sulfoxide derived from naphthyl methyl sulfide (4f). Thus, CiP may be utilized for the asymmetric synthesis of optically active hydroperoxides 1, alcohols 2, and sulfoxides 5.

Journal Article↗
PubMed2002

Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus. Alternative truncation and expression in meiosis.

The primary purpose of the present study was to investigate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete, Coprinus cinereus. Interestingly, although CoPCNA is a single-copy gene in the genome, two different PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated as CoPCNA-alpha and CoPCNA-beta. CoPCNA-beta was made by truncating at specific sites in CoPCNA-alpha mRNA, 5'-AAGAAGGAGAAG-3' and 5'-GAAGAGGAAGAA-3'. Both of these sequences were present in exon IV in the genomic sequence, and interestingly the former was the same as the inverse sequence of the latter. CoPCNA-alpha was 107 amino acids larger than human PCNA, and so the 107 amino-acid sequence was inserted in a loop, the so-called D2E2 loop, in human PCNA. Northern blotting analysis indicated that CoPCNA was expressed not only at premeiotic S but also at the meiotic prophase stages such as leptotene and early zygotene, just before and when karyogamy occurs and the homologous chromosomes pair. Western blotting analysis using anti-(CoPCNA-alpha) Ig revealed that at least two CoPCNA mRNAs before and after truncation were translated at the meiotic prophase as CoPCNA-alpha and CoPCNA-beta.

Basidiomycota↗
PubMed1974

The effect of light on fruiting body formation and adenosine 3':5'-cyclic monophosphate metabolism in Coprinus macrorhizus.

The monokaryotic mycelia of a mutant strain, fis(c), of Coprinus macrorhizus, which are able to form monokaryotic fruiting bodies in the light, failed to form any fruiting bodies in darkness. A dikaryon and a mutant strain, ds, formed malformed fruiting bodies in darkness. Illumination for 1 day of fis(c) mycelia grown in darkness for 4 days or longer was effective in inducing malformed fruiting bodies. The accumulation of adenosine 3':5'-cyclic monophosphate in the illuminated mycelia of strain fis(c) was demonstrated. The illuminated mycelia of strain fis(c) produced high levels of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), which degrades cAMP, while the dark-grown mycelia showed no or very low activities of these enzymes. Dikaryotic mycelia and monokaryotic mycelia of strain ds produced significant amounts of these enzymes even in darkness. When the dark-grown mycelia of strain fis(c) were exposed to continuous light, the activities of adenylate cyclase and phosphodiesterase increased rapidly after a lag period whose length depends on the culture age of mycelia. Cycloheximide inhibited the increase in these enzyme activities stimulated by light. When the fis(c) mycelia were exposed to continuous light, an increase in cAMP-binding activity was observed. A possible participation of cAMP in the formation of fruiting bodies in C. macrorhizus is discussed.

Adenylyl Cyclases↗
PubMed1992

The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus.

The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical A alpha and A beta loci. Four genes, one alpha and three beta, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes.

Alleles↗
PubMed1995

An N-Terminal Dimerization Domain Permits Homeodomain Proteins To Choose Compatible Partners and Initiate Sexual Development in the Mushroom Coprinus cinereus.

The A mating-type locus of the mushroom Coprinus cinereus contains three or more paralogous pairs of genes encoding two families of homeodomain proteins (HD1 and HD2). A successful mating brings together different allelic forms of at least one gene, and this is sufficient to trigger initial steps in sexual development. Previous studies have suggested that development is regulated by heterodimerization between HD1 and HD2 proteins. In this report, we describe 5[prime] gene deletions and 5[prime] end exchanges showing that the N-terminal regions of the proteins are essential for choosing a compatible partner but not for regulating gene transcription. Using an in vitro glutathione S-transferase association assay, we demonstrated heterodimerization between HD1 and HD2 proteins and found that heterodimerization only occurs between compatible protein combinations. The N-terminal regions of the proteins were sufficient to mediate dimerization, and N-terminal swaps resulted in a predicted change in dimerization specificity. By analyzing the N-terminal amino acid sequences of HD1 proteins, we identified two potential coiled-coil motifs whose relative positions vary in paralogous proteins but are both required for in vivo function.

Journal Article↗
PubMed1997

Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus.

The laccase from the fungus Coprinus cinereus has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small plate-like crystals of an enzymatically deglycosylated form of the enzyme have been grown by the hanging-drop method using polyethylene glycol as precipitant. These crystals diffract to at least 2.2 A. They belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.4, b = 85.7, c = 143.1 A with a single molecule of laccase in the asymmetric unit.

Journal Article↗
PubMed1969

Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller).

The occurrence and properties of enzymes of carbohydrate metabolism were studied during dikaryotic fruiting of the mushroom Coprinus lagopus. Enzymes of hexose monophosphate catabolism, sugar alcohol (polyol) dehydrogenases (DH), and trehalase occurred throughout development. The ratio of xylitol DH to sorbitol DH was greater than unity in both monokaryotic mycelium and dikaryotic fruit body caps, whereas this ratio decreased in the stipe (stalk) tissue. Xylitol DH and sorbitol DH were both dependent upon nicotinamide adenine dinucleotide (NAD) and showed maximal activity at pH 9. Two separate enzymes were suspected on the basis of preferential utilization of the NAD analogue, thionicotinamide-NAD, by xylitol DH, and this feature was consistent throughout development. An appraisal of the carbohydrate pool revealed trehalose and glucose, with the former predominant in the stipe and the latter in excess in the cap of dikaryotic fruit bodies. Trehalase activity in dialyzed enzyme extracts showed pH optima at acid and alkaline pH levels in monokaryotic mycelium, dikaryotic stipes, and cap tissues.

Alcohol Oxidoreductases↗
PubMed1973

Purification and identification of the fruiting-inducing substances in Coprinus macrorhizus.

Substances which are effective in inducing fruiting bodies in monokaryotic mycelia of the fis(+) strain of Coprinus macrorhizus were purified and characterized. The active components of fruiting-inducing substances were identified as adenosine-3'-monophosphate, adenosine 3',5'-cyclic monophosphate (cyclic AMP), and a protein which is bound with the cyclic AMP. Cyclic AMP was synthesized from adenine within mycelia of the mutant strains which form monokaryotic fruiting bodies without the addition of fruiting-inducing substances, but not in those of the strains which do not form monokaryotic fruiting bodies. The proteins which bind with cyclic AMP were detected in crude extracts of mycelia of those strains which form monokaryotic fruiting bodies and of the dikaryon, but not in those of the strains which do not form monokaryotic fruiting bodies.

Adenine↗
PubMed1973

Metabolism of adenosine 3',5'-cyclic monophosphate and induction of fruiting bodies in Coprinus macrorhizus.

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.

Adenosine Monophosphate↗
PubMed1974

Effect of glucose on the fruiting body formation and adenosine 3',5'-cyclic monophosphate levels in Coprinus macrorhizus.

The formation of fruiting bodies in the monokaryotic fis(c) strain and a dikaryon of Coprinus macrorhizus was inhibited by growth in high-glucose media. In high-glucose media the characteristic burst of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation during fruiting-body formation was absent. Enzymatic activity assays revealed that mycelia grown in high-glucose media contained relatively lower amounts of adenylate cyclase and cAMP-phosphodiesterase than mycelia grown in low-glucose media. The synthesis of inducible d-serine deaminase and tryptophanase was repressed in high-glucose media. A mutant (gluR) in which the glucose repression of fruiting-body formation is affected was isolated by selection in high-glucose media. The mutation caused the cAMP levels to be no longer affected by glucose and affected ability to synthesize the inducible d-serine deaminase and tryptophanase. The gluR mutant was partially dominant in dikaryons. It is suggested that cAMP may play important roles in inducing fruiting bodies and in controlling inducible enzyme synthesis in C. macrorhizus.

Adenylyl Cyclases↗