| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
1) Cell wall glucans of Schizosaccharomyces pombe were isolated by combined use of sodium hydroxide extraction and enzymatic digestion. By metaperiodate oxidation, ^<13>C-NMR spectroscopy and ^<13>C-CP-MAS spectroscopy, their structures were found to beα-1,3-, β-1,3-, and β-1,6-glucans. The β-1,6-glucan was highly branched at C-3 by β-glucopyranoside residue to form a comb-shape structure. In contrast, the β-1,6-glucan in Candida albicans and Saccharomyces cerevisiae had only a few branches. The signals of α-1,3-glucan and β-1,3-glucan were shown on ^<13>C-CP-MAS spectra. This result suggested that these glucans had high crystallinity and form the framework of the cell wall.
2) We prepared antibodies (Ab) againstα-1,3-glucan or highly branched β-1,6-glucan isolated by 1) in rabbits.
3) A high pressure freeze-substitution method was developed for immunoelectron microscopy (immuno-EM) of S.pombe. The method used a low concentration of OsO_4 or uranyl acetate (UA) provided a high contrast i
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n the specimen, while retaining the fine structure of the cells. In the image fixed with 0.01% OsO_4-acetone, the cell wall and cytoskeleton were visible, while 0.1% UA-acetone was superior in the membranous structure and cytoplasm. The new method makes it possible to visualize the ultrastructure and localization of the material on one thin section for the immuno-EM.
4) Specificity and usefulness of these antibodies were examined on the samples cryofixed by high-pressure-freezing described 3). α-1,3-glucan was observed on the surface of the cell wall and the region adjacent to the cell membrane. In contrast, highly branched β-1,6-glucan was located through out the glucan layer and was linear arranged radiating from the cell membrane.
5) Glucan biosynthesized in the cell free system formed amorphous particles. These particles stretched into fine fibrils (2 nmφ) and, arranged in parallel, developed into ribbon-shaped bundles. The glucan grew straight, but did not form a network. The process of glucan biosynthesis of cps8 mutant was the same as wild type, unlike the protoplast regeneration system. These results clearly showed that the cell wall substance and regulated factors transported from the cytoplasm were essential for the glucan network formation. Less
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