White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.     

An endo-exonuclease from meiotic tissues of the basidiomycete Coprinus cinereus. Its purification and characterization.

An endo-exonuclease has been identified and partially purified from the basidiocarp tissues of the basidiomycete Coprinus cinereus, which include synchronous meiosis at karyogamy-pachytene stages. Its peak activity appears during the meiotic prophase. The Coprinus endo-exonuclease has a single-strand specific endonuclease activity that converts the supercoiled DNA to relaxed DNA. The endonucleolytic cleavage of single-strand DNA generates 3'-phosphomonoester termini. It is also a single-strand-specific exonuclease and it hydrolyzes linear DNA in a 3' to 5' direction, but is unable to hydrolyze single-strand DNA having a 3'-phosphomonoester terminus. It requires Mg2+ with an optimal concentration of 25 mM. It has an optimal pH of 8.3, a peak enzyme activity at 50 degrees C, and it contains a single 43-kilodalton polypeptide. Coprinus meiotic endo-exonuclease may be involved in the substrate preparation for meiotic recombination.     

Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.     

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

Ubiquitin immunoreactivity shows several proteins varying with development and sporulation in the basidiomycete Coprinus cinereus

Crude extracts of mycelia and basidiocarp primordia in the basidiomycete Coprinus cinereus were resolved on sodium dodecyl sulfate--polyacrylamide gels, and ubiquitin and several proteins were detected by immunoblotting with anti-ubiquitin antibody. The molecular masses of the proteins detected were 30,900, 28,600, 27,800, 26,300, 22,500, and 15,400 daltons, respectively. Relative levels of ubiquitin and most of the ubiquitin-immunoreactive proteins in basidiocarp primordium formation increased and in basidiocarp maturation decreased in cap and upper stipe, while in lower stipe became high except for the 27,800 dalton protein and ubiquitin. During sporulation, ubiquitin and all the ubiquitin-immunoreactive proteins tended to decrease in the cap of the young wild-type basidiocarp. The levels of 30,900 and 15,400 dalton proteins increased transiently at 6-10 h after the beginning of the last light period, while ubiquitin decreased markedly. No correlation was observed between changes in levels of the ubiquitin-immunoreactive proteins and the blocked stages in sporulation-deficient mutants.     

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

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.     

Purification and characterization of an ubiquitin-immuno-reactive protein localized in the cap of young basidiocarp in the basidiomycete Coprinus cinereus.

The ubiquitin-immuno-reactive protein with a molecular weight of 27,800 daltons, which is mainly present in the cap of young basidiocarp, was purified from the basidiomycete Coprinus cinereus. The molecular weight of the native protein was approximately 55,000 as determined by gel filtration chromatography. The isoelectric point of the protein was 4.4. The amino-terminal sequence of the protein was also determined.     

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

Inheritance of DNA methylation in Coprinus cinereus.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.     

Glutamate metabolism in a monokaryon of Coprinus cinereus.

The possible involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation is reported in a monokaryon of Coprinus cinereus.     

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.     

Molecular breeding of the basidiomycete Coprinus cinereus strains with high lignin-decolorization and -degradation activities using novel heterologous protein expression vectors

Two chromosome-integrating vectors, pLC1 and pLC2, were used. The former is the pUC19-based vector carrying the Lentinus edodes ras gene promoter and priA gene terminator, and the latter is the pBR322-based vector carrying the promoter and terminator of the priA gene. The manganese (II) peroxidase (MnP) cDNA (mnpc) derived from Pleurotus ostreatus was fused between the promoter and terminator of pLC1 and pLC2, yielding the recombinant plasmids pLC1-mnp and pLC2-mnp. These plasmids were introduced into protoplasts of the Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid pCc1001 by co-transformation. Two Trp⁺ transformants for each plasmid, showing clearly higher lignin-decolorization activities, were obtained through introduction of pLC1-mnp and pLC2-mnp. Southern-blot analysis revealed that the four transformants all possess the mnpc sequence on their chromosomes. One Trp⁺ MnP⁺ transformant (named TF2-7), which was derived from the introduction of pLC2-mnp and carried the highest number of copies (approx. 10) of mnpc, showed remarkably high lignin-decolorization and -degradation activities; at the time of cultivation when only 35%–40% of the lignin was decolored and degraded by the control Trp⁺ transformant obtained by the introduction of pCc1001 alone, almost all of the lignin was decolored and degraded by TF2-7. Received: 20 October 1997 / Accepted: 31 October 1997