Growth regulation inCoprinus radiatus

Some of the morphological and physiological parameters of stipe growth or elongation inCoprinus radiatus were investigated. During the development of the fruit body the number of cells in a row in the growing portion of the stipe doubled during the development of the button, and again during the phase of rapid stipe elongation. Also during the stage of rapid elongation the cells in the upper 2/3 of the stipe increased 6–8 fold in length. The existence of a growth regulator synthesized in the cap and exerting control over the stipe was demonstrated through decapitation experiments. The cap appears to be required for normal stipe development until the stipe reaches about 1/4 of its final length. Through decapitation and cap-stipe exchanges it was found that the cap produced growth regulator up to the time of autodigestion; however, the stipe responded to the regulator only during a brief period at the onset of elongation.     

Behavior of chromosomes after meiosis inCoprinus cinereus

The movement was investigated of a specific chromosome in the F₁ progeny of the basidiomyceteCoprinus cinereus. We focussed our attention on the smallest chromosome of the 5302 strain. We first constructed a chromosome-smallest library and screened it with a chromosome-specific clone, pRC 1. The pRC 1 probe hybridized only with the smallest chromosome of the 5302 strain, and it detected one band of different mobility in two parental strains. In the F₁ progeny, the probe hybridized with one to three chromosomes. Most of the hybridized chromosomes in the F₁ progeny were positioned in terms of mobility between the hybridized chromosomes of the two parental strains. Therefore, they were probably generated by meiotic recombination between homologous chromosomes of different sizes.     

Partial contig map of the smallest chromosome inCoprinus cinereus

We have constructed a chromosome-specific cosmid library from electrophoretically separated chromosomes of the basidiomyceteCoprinus cinereus and performed contig mapping and analysis of chromosome length polymorphisms (CLPs) for the smallest chromosome of the 5302 strain. A contig map of about 300 kb indicated that the novel size chromosomes in the F₁ progeny were apparently recombinants containing physical markers derived from both ends and central regions in this map. This may be the first case in which the formation of CLPs in the F₁ generation has been explained using the contig map. The results obtained were consistent with the hypothesis that novel CLPs were produced by meiotic recombination between the parental homologous chromosomes of unequal sizes.     

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

Darkness-induced factor affecting basidiocarp maturation in Coprinus macrorhizus

Graftings were made between young basidiocarps of Coprinus macrorhizusgrown under different light programs. Results showed that adiffusible factor(s) affecting basidiocarp maturation was inducedby the darkness of phase 3. (Received July 3, 1979; )     

Photo-induced karyogamy in a Basidiomycete, Coprinus macrorhizus

In Coprinus macrorhizus, the fruit-body matured when two 2-hrlight periods were given separately after the formation of theprimordium. The karyogamy may be triggered by the exposure tothe second light, because fusion of two nuclei in the basidiumwas always observed 12 hr after exposure to the second light,irrespective of the time when given. (Received August 15, 1973; )     

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.     

Cytological studies on sporeless mutant in the basidiomyceteCoprinus macrorhizus

Meiotic events in the sporeless mutant, NG107, and in the wild-type dikaryon of the basidiomyceteCoprinus macrorhizus were studied comparatively with light and electron microscopes in order to determine precisely which meiotic process was blocked in the mutant. In most basidia of the mutant, when incubated at 28 C, meiosis stopped at meta-anaphase I although the kinetic apparatus was formed. Then, the basidia became mononucleate, electron dense, and finally disintegrated. This mutant, when incubated at 20 C, produced basidiospores approximately one third of the wild-type. Basidia with irregular number of spores were observed in higher frequency than in the wild-type. Experimental results of temperature shifts from 28 C to 20 C and the reciprocal shift suggest that meta-anaphase I might be thermosensitive in the mutant.     

Effects of light on basidiocarp maturation in Coprinus macrorhizus

The effect of white light on basidiocarp maturation in Coprinusmacrorhizus was examined. The results obtained showed that basidiocarpmaturation was separable into five successive phases in regardto photosensitivity: phase 1 in which light triggered maturation;phase 2 in which light delayed maturation; phase 3 in whichlight stopped maturation; phase 4 in which light acceleratedmaturation; and phase 5 in which light had no effect upon theprogress of maturation. The relation between the light effect and the events duringspore formation including nuclear fusion, meiosis and sterigmaformation was examined with the following results: the lightof phase 1 initiated nuclear fusion; the light given in phase2 delayed the initiation of nuclear fusion and the later progressup to prophase 1; without the darkness of phase 3, meiosis neverproceeded beyond prophase 1; and the light given in phase 4accelerated the progress from the initiation of nuclear fusionto prophase 1. (Received August 15, 1977; )     

Effect of cyclic AMP on glycogen phosphorylase in Coprinus macrorhizus.

Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransfase, EC 2.4.1.1) activity was found in mycelial extracts of Coprinus macrorhizus concurrently with decrease of glycogen content in mycelial cells. Incubation of the enzyme sample with cyclic AMP and ATP leads to a 3-fold activation of the glucogen phosphorylase activity. Activation of the enzyme partially purified through Sepharose 6B required a cellular fraction containing cyclic AMP-dependent protein kinase.     

Effects of amino-acid substitutions in β tubulin on benomyl sensitivity and microtubule functions inCoprinus cinereus

The sensitivity of the homobasidiomyceteCoprinus cinereus to the benzimidazole fungicide benomyl allowed us to isolate β-tubulin mutants as strains resistant to benomyl. To understand the molecular basis for the interaction between benomyl and β tubulin and for cellular defects in the β-tubulin mutants, we first analyzed the wild-type β1-tubulin gene (benA) ofC. cinereus, revealing thatbenA contains eight introns and encodes a 445 amino-acid protein. We then characterized 16 β1-tubulin mutants. The 16 mutations involved 11 different amino-acid substitutions at 10 different residues in β1 tubulin. The mutated residues were widely distributed along the primary sequence of β1 tubulin, from residue 3 in the N-terminal domain to residue 350 in the intermediate domain, but half of them appeared to be close to the αβ intradimer interface in an atomic model determined by electron crystallography. The benomyl resistant strain BEN 193, which exhibits clear heat sensitivity for hyphal growth and defects in various cellular processes, had a novel mutation, i.e., the Leu to Phe substitution at residue 350. Benomyl resistance and the heat sensitivity in BEN 193 were suppressed by additional amino-acid substitutions at various residues in β1 tubulin, suggesting that conformational changes of β1 tubulin are involved in the alterations. The DDBJ/GeneBank/EMBL accession number for the sequence reported in this paper is AB000116.     

Isolation and genetic analysis of resistant mutants to the benzimidazole fungicide benomyl inCoprinus cinereus

In total, 404 variants resistant to the antimicrotubule agent benomyl were isolated from UV-irradiated oidia of the basidiomyceteCoprinus cinereus. Part of the variants showed, in addition to benomyl resistance, heat sensitivity or heat dependence. Fifteen variants selected on the basis of different phenotypes were subjected to further analyses. All of the 15 variations were due to single-gene mutations, and the mutations comprised four groups (benA, benB, benC, andbenD) in terms of genetic linkage. Some of the 15 mutations affected nuclear migration in dikaryosis and/or fruiting processes.     

Effects of light on fruit-body formation in a basidiomycete, Coprinus macrorhizus

A basidiomycete, Coprinus macrorhizus produced only vegetativemycelia, when cultured under continuous darkness. Under continuouswhite light, visible tiny primordia formed on the 6th day afterinoculation, followed by normal development to fruit-bodies.Spores disseminated on the 11th day. However, when cultureswhere transferred to continuous darkness after the formationof primordia, rudimentary pilei with slender stalks formed.Abundant hairy hyphae were produced along the entire surfaceof the slender stalks. Thus, light was required-for both theinitiation and development of the fungal fruit-body. A fairy ring of primordia formed along the narrow region ofmycelia formed just prior to the exposure to light, provideda colony was pre-grown for more than 4 days in the dark, thenexposed to light for 24 hr or longer. Exposure to light for at least 48 hr during the period between24th and 96th hr after primordium initiation was required fornormal maturation of the fruit-body. This 48 hr light periodneeded for maturation of the fruit-body could be substitutedby two, 2 hr light periods given at the beginning and the endof the 48 hr period. We have tentatively concluded that lightis required at two definite stages for fruit-body developmentafter formation of the primordium. Effective wavelengths for both the initiation and developmentof fruit-bodies were in the near ultraviolet and blue regions. (Received July 24, 1972; )     

Basidiospore formation in monokaryotic fruiting bodies of a mutant strain of Coprinus macrorhizus

The process of basidiospore formation in a mutant strain Fis^c of Coprinus macrorhizus, a heterothallic species of Basidiomycete, which forms monokaryotic fruiting bodies was examined. A single nucleus in a young basidium divided mitotically and two daughter nuclei were fused subsequently. The fused nucleus then divided meiotically forming four basidiospores on a basidium. The typical chromosome behaviours in the first meiotic prophase were observed. Synaptonemal complexes were observed in a basidium at the first meiotic prophase. A continuous illumination of fruiting bodies was effective to arrest meiosis in monokaryotic fruiting bodies at the particular stage of meiotic division.     

Adenosine 3',5'-monophosphate-receptor protein and protein kinase in Coprinus macrorhizus

A single cAMP-receptor protein could be detected in mycelial extracts of Coprinun macrorhizus by using the photoaffinity cAMP-analogue, 8-N3-cAMP. The protein which specifically bound 32P-labeled 8-N3-cAMP had an apparent molecular weight of 46,000 as determined by an SDS-polyacrylamide gel electrophoresis system. The 46,000-dalton protein was characterized by the dissociation constant for [32P]-8-N3-cAMP, and by the nucleotide specific inhibition of [32P]-8-N3-cAMP binding. The 46,000-dalton protein was co-chromatographed on a DEAE-cellulose column with cAMP-dependent protein kinase. The levels of [32P]-8-N3-cAMP-binding and protein kinase activities in mycelial extracts of strains used was always in parallel. The result indicated that the 46,000-dalton protein may be a regulatory subunit of protein kinase with the capacity to bind cAMP. cAMP-dependent protein kinase of this fungus was immunologically different from those of higher animals.     

Sequential production of enzymes and basidiospore formation in fruiting bodies of Coprinus macrorhizus

The production of NADP-dependent glutamate dehydrogenase was initiated at the stage of first meiotic prophase in pileus cells but not in stipe cells of dikaryotic and monokaryotic fruiting bodies in Coprinus macrorhizus. The production of chitinase and glucanases assayed with laminarin and lichenan was observed after the completion of meiosis only in pileus cells. The light conditions that were effective for the delay or inhibition of cellular events in the pileus cells were also effective for the delay or inhibition of enzyme production. But all sporeless mutants tested, which were defective at the various stages of basidiospore formation, produced the normal levels of these enzymes. The results indicate that the sequential production of enzymes and cellular events leading to basidiospore formation in pileus cells are independent from each other.Abbreviation GDH_NADP NADP-dependent glutamate dehydrogenase