Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene.

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp(-)) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.

Isolation of novel bacteria and actinomycetes using soil-extract agar medium.

Novel bacteria were discovered using an isolation technique consisting of (i) selection of microorganisms that grew on soil-extract agar medium, but not on conventional media, and (ii) detection of small microbial colonies with a microscope. Three bacterial strains thus isolated were provisionally designated Shinshu-th1, -th2, -th 3, and five actinomycete strains, Shinshu-MS-01, -02, -03, -04, -05, respectively. Sequence analysis of their 16S rDNA showed that th1 had 95--96% homology with three unculturable bacteria, and th2 had 96% similarity to Bradyrhizobium sp., one unculturable and one unidentified bacterial strain. A phylogenetic study indicated that both strains were alpha-Proteobacteria belonging to the order Rhizobiales and the family Bradyrhizobiaceae. Since they had low homology (96%) with their close relatives, it is possible that th1 and th2 belong to a new genus. The actinomycetes Shinshu-MS-02 and -03 had 95--96% homology with four strains of Actinomadura, -04 had 95--96% similarity to Streptosporangium and Microbispora, and -05 had 97--98% homology with three strains of Acrocarpospora, Herbidospora and Planotetraspora. According to the phylogenetic study, both 02 and 03 are possibly new species of Actinomadura, -04 of Streptosporangium, and -05 of Acrocarpospora. Shinshu-th 3 and -MS-01 were identified as Mycobacterium cookii and Frankia sp., respectively, having 99% homology with these species.

Rare bacterium of new genus isolated with prolonged enrichment culture.

Dynamic change in microbial flora was monitored with an oxygen electrode. The 1st phase microorganisms, which first grew well in LB medium, were followed by the 2nd phase microorganisms, which supposedly assimilated microbial cells of the 1st phase and their metabolites. In a similar way, a change in microbial flora was observed from the 1st phase to the 4th phase in 84 hr. Based on this observation, prolonged enrichment culture was done for as long as two months to increase the ratio of existence of rare microorganisms. From these culture liquids, four slow-growing bacteria (provisionally named Shinshu-ah1, -ah2, -ah3, and -ah4), which formed scarcely visible small colonies, were isolated. Sequence analysis of their 16S rDNA showed that Shinshu-ah1 had 97% homology with Bradyrhizobium japonicum and uncultured alpha proteobacterium clone blaii 16, Shinshu-ah2 91% with Rasbo bacterium, Alpha proteobacterium 34619, Bradyrhizobium genosp. P, Afipia felis and an unidentified bacterium, Shinshu-ah3 99% with Methylobacterium mesophilicum, and Shinshu-ah4 95% with Agromyces ramosus DSM 43045. Phylogenetic study indicated that Shinshu-ah2 had a possibility to form a new family, Shinshu-ah1 a new genus, and Shinshu-ah4 a new species.

Effects of indole and caffeine on cAMP in the ind1 and cfn1 mutant strains of Schizophyllum commune during sexual development.

The ind1 and cfn1 mutations of Schizophyllum commune express resistance to high concentrations of indole and caffeine respectively, and also affect sexual development. To clarify molecular events caused by the mutations, it was investigated how cAMP levels in S. commune strains respond to externally supplied indole and caffeine. Both compounds increased the cAMP levels in wild-type strains under several culture conditions. During sexual development of the ind1 mutant, the cAMP level in an early stage (hyphal aggregation) was highly increased by addition of indole, and the phenomenon disappeared in a later stage (fruit body formation). For the cfn1 mutants, the incremental increase in cAMP levels by addition of caffeine was smaller than that of wild-type strains.

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor).

Recently the mesoderm-inducing effects of the transforming growth factor ß (TGF-ß) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF-ß family has mesoderm-inducing ability.

Activities of Mesoderm-Inducing Factors Secreted by Mammalian Cells in Culture1 : (mesoderm-induction/activin A/EDF/TGF-ß family/embryonic induction).

We have examined the activities of several mesoderm-inducing factors contained in the culture fluids of phorbol ester (4beta-phorbol 12-myristate 13-acetate;PMA)-stimulated human cell lines. Mesoderm induction was assayed by examining the differentiation of mesoderm tissues reacted with presumptive ectoderm of the Cynops blastula. The assay system also examined erythroid differentiation activity (EDF activity) in order to test the relationship between mesoderm induction and activin A (EDF). Of 22 human cell lines examined, six strains were positive for both mesoderm-inducing activity and EDF activity. Four strains showed only mesoderm inducing activity, and one showed only EDF activity. The remaining 11 strains showed neither activity. Therefore, most cell lines secreting mesoderm-inducing activity also possessed EDF activity. Furthermore, culture fluid of a strain (K-562) that exhibited both types of activities, was partially fractionated by DEAE-Toyopearl column chromatography and examined in the same way. The fractions that showed the highest amount of EDF activity were coincident with those displaying mesoderm-inducing activity. These results suggest that a number of PMA-stimulated mammalian cell lines have the ability to secrete mesoderm-inducing factors which are similar to activin A (EDF).

Significance of an Increase of Intracellular Adenosine Concentration for Dormancy in Starfish Blastulae: (starfish embryos/blastula/adenosine/dormancy).

Adenosine at concentrations greater than 6 µg/ml halted embryonic development of the starfish Asterina pectinifera specifically at the 256-cell stage which corresponds to the onset of blastulation. When a fertilized egg was cultured continuously in sea water containing adenosine from fertilization, a gradual increase in intracellular concentrations of free adenosine was observed before a cessation of development took place. On the other hand, intracellular concentrations of ATP, ADP and AMP in the embryo cultured in sea water containing adenosine were nearly the same as those of an embryo cultured in sea water without adenosine. By returning the development-arrested embryo to normal sea water the embyro developed normally to the bipinnaria stage accompanied by a gradual decrease in the intracellular cencentration of adenosine. Treatment of fertilized eggs with 9-ß-d-arabinofuranosyl-9H-purine-6-amine (25 µg/ml) or 2'-deoxyadenosine (10 µg/ml) halted development specifically before the onset of blastulation in an irreversible manner. Embryos treated with 3'-deoxyadenosine (50 µg/ml) shortly after fertilization developed to healthy blastulae but hatching never occurred. These results exclude the possibilities that the action of adenosine is mediated by the inhibition of DNA synthesis or RNA polyadenylylation.

Development of Fertilized Starfish Eggs in Which Cytokinesis is Prevented by Iturin A-2: (starfish embryos/iturin A-2/cytokinesis/blastulation/1-methyladenine).

Iturin A-2, a cyclic peptide obtained from cultures of Bacillus subtilis inhibited cell division but not nuclear divisions of fertilized eggs of the starfish Asterina pectinifera, resulting in the formation of non-cleaving eggs containing all the embryonic nuclei in a common cytoplasm. Fertilized eggs in which cleavage was prevented by iturin A-2 (50µg/ml) synthesized DNA, RNA and protein at comparable rates to those of normal embryos up to the onset of blastulation. In single-cell embryos, however, elevation of the level of transcription, which is a characteristic marker of blastulation, did not take place and the chromatin masses eventually dispersed in the cytoplasm. Treatment of oocytes with iturin A-2 (50µg/ml) resulted in loss of their response to the maturation-inducing substance 1-methyladenine, whose receptors are located in the cell membrane or other components of the oocyte cortex. Furthermore, iturin A-2 bound to oocyte cortices quantitatively, suggesting that its site of action is the cortices of oocytes and eggs, and that its arrest of cleavage of fertilized eggs is due to loss of the ability of the cell membrane to form a cleavage furrow.