In our previous study, the plasma-polymerization rapid-freeze replica(PPRFR)method has been developed for observing biological specimens in a natural state and at high resolution by transmission electron microscopy(TEM)(Yamaguchi et al., 1997). In this method, more faithful three-dimensional ultrastructure is copied on the inner side of the replica film, and the replica film is amorphous in texture and hard enough to withstand against probe force of the tip. Therefore, it is expected to get even higher resolution of the specimen images if the PPRFR film is scanned on the specimen side by atomic force microscopy(AFM). In the present study, the PPRFR film of T4 bacteriophages were observed by AFM, and the images were compared with those obtained by TEM. Conventional metal freeze-replica of spores of a fungus Cladosporium sp. was also examined by AFM, and compared with TEM images. This is the first report that PPRFR was applied to AFM.

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The homobasidiomycete Coprinus cinereus is easy to grow in the laboratory and produces fruit bodies in a relatively short period(about 10 days). The fungus is heterothallic, undergoes remarkable morphogenesis as well as naturally synchronous meiosis in the sexual process, and hence provides a unique opportunity for studies of sexuality, meiosis and morphogenesis. To elucidate the molecular mechanisms for sexuality and morphogenesis in C. cinereus, we have been analyzing various mutants having defects in the processes.

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The development of perithecia in Neurospora crassa is under the control of light, nutritional condition such as nitrogen starvation and temperature. Blue light can stimulate(i)the formation of protoperithecia on nitrogen deficient media, (ii)positive bending of perithecial beak, and(iii)the ejection of ascospores from asci in the perithecia. We found that the position of the formation of perithecial beak was dependent on light.(iv)Light stimulated the positioning of perithecial beak on the upperward of it, which we defined as“ light induced polarity of perithecia”. We developed in vitro systems to analyse molecular mechanism of light signal transduction that used the membrane and soluble fractions of mycelial extract. UV-A light induced the binding of ATP/GTP to 58kDA, 77kDa, 83kDa, and 129kDa proteins in the membrane fraction. Blue light stimulated the ADP-ribosylation of 38kDa and 56kDa proteins in the soluble fraction. Blue light also stimulated the phosphorylation of 15kDa protein in the membrane fraction. We isolated a mutant missing completely the phosphorylation of 15kDa protein in the membrane fraction. The mutation designated as psp(phosphorylation of small protein)was mapped on LGVR between al-3and his-6. The psp mutats showed blindness in the light induced polarity of perithecia. The 15kDa protein was purified and the cDNA was cloned. It was identified as nucleoside diphosphate(NDP)kinase. The cDNA for NDP kinase prepared from psp mutants included a mutation of amino acid replacement from Pro 72to His. The putative function of NDP kinase in the light signal transduction to perithecial morphogenesis was discussed.

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The fission yeast Schizosaccharomyces pombe grows at the cell ends .F-actin was found to be localized at the cell ends, and correlated with polarized growth of the cell wall.Our studies by three-dimensional reconstruction of serial sction electron microscopy demonstrated the presence of vesicles and filasomes at the cell ends. Growth arrest in distilled water and cytochalasin treatment caused the remarkable changes of actin distribution as well as cytoplasmic organelles. These studies indicated the close relationship between actin and cell wall formation in the polarized growth. Recent studies on tip growth in filamentous fungi and on the morphological mutants in fission yeast were reviewed.

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Summary: Plastid-dividingrings have been observed at the isthmus region of the dividing plastids in the wide range of land plants and algae, and have been envisaged as a division apparatus of plastids. As proteins composing the ring are suggested to be nulear-encoded, the mechanism of plastid divislon may have been aquired during the course of the evolutlon from cyanobacterium-like. endosymbionts to true organelles, i. e. plastids. ln most of the land plants examined so far and some green algae, the plastid-dividing rings are detected as double ring structures girdling the double envelope; the outer cytoplasmic ring and the inner stromal ring. In the other green algae and red algae, a single cytoplasmic ring has been recorded. The evidence now encompasses those of chromophytic algae of which chloroplasts are surrounded by four membranes. Their plastid dividing ring is observed as a single ring girdling the periplastidal face of the outer membrane of the inner pair of the four surrounding membranes. This finding demonstrates that the inner pair represents the true chloroplast envelope, providing a new evidence supporting the secondary endosymbiosis theory for the origin of the chloroplasts of chromophyta. Discussions are made on themorphology and localizations of plastid-dividing rings in relevance to the origins and evolution of plastids.

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Fertilization of red algae is achieved by fusion of a spermatium and a trichogyne, the specialized process of a carpogonium(egg cell). Recently, studies of red algal fertilization have been carried out using species suitable for detailed observations and experimental research. These studies revealed ultrastructural and histochemical properties of spermatium and trichogyne that are responsible for the specific attachment between non-motile gametes during fertilization. The process of the fertilization also includes some cellular events, i. e., resumption of spermatial nuclear division and cell wall formation. These events are induced by gamete attachment and followed by cytoplasmic and nuclear fusion between gametes. There are significant variations in the morphological and histochemical features of gametes and processes of fertilization among red algae. To understand how such considerable variety of fertilization mechanisms has occurred during evolution of red algae, detailed, comparable information in a wide range of taxa is necessary.

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We have analyzed the DNA sequences of the mitochondrial cytochrome oxidase subunit l(COX)gene in algae, We found the deduced deviant genetic codes where UAG codes for alanine or leucin e in Chlorophyceae, UGA codes for tryptophan in Prymnesiophyceae and AUAcodes for methionine in Xanthophyceae. Examination of the mitochondrial genetic codes in algae appears to be a very useful genetic marker for classification of algae when it is considered with the results obtained through a molecular phylogenetic tree. By using both approaches with a molecular maker and a morphological maker, the relationship among algal species will be confirmed.

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Hagemann and Gleissberg(1996)Proposed the new term“the blastozone” .From the standpoint of the idea of the blastozone, lt is needed to investigate whether the morphogenesis induces the histogenesis or not, and the morphogenesis should be emphasized in the study of the higher plants.

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