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To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze- substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. 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Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae by using the affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase
http://hdl.handle.net/10091/197
http://hdl.handle.net/10091/197cf90f995-a949-4bc7-8bef-1832241e49d5
名前 / ファイル | ライセンス | アクション |
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Direct labeling of polyphosphate.pdf (707.0 kB)
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Item type | 学術雑誌論文 / Journal Article(1) | |||||
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公開日 | 2007-05-07 | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae by using the affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase | |||||
言語 | ||||||
言語 | eng | |||||
DOI | ||||||
識別子タイプ | DOI | |||||
関連識別子 | https://doi.org/10.1128/AEM.71.10.5692-5701.2005 | |||||
関連名称 | 10.1128/AEM.71.10.5692-5701.2005 | |||||
資源タイプ | ||||||
資源 | http://purl.org/coar/resource_type/c_6501 | |||||
タイプ | journal article | |||||
著者 |
Saito, K
× Saito, K× Ohtomo, R× Kuga-Uetake, Y× Aono, T× Saito, M |
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信州大学研究者総覧へのリンク | ||||||
氏名 | Saito, K | |||||
URL | http://soar-rd.shinshu-u.ac.jp/profile/ja.HCkpPUkh.html | |||||
出版者 | ||||||
出版者 | American Society for Microbiology | |||||
引用 | ||||||
内容記述タイプ | Other | |||||
内容記述 | Applied and Environmental Microbiology. 71(10): 5692-5701 (2005) | |||||
書誌情報 |
Applied and environmental microbiology 巻 71, 号 10, p. 5692-5701, 発行日 2005-10 |
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資源タイプ(コンテンツの種類) | ||||||
内容記述タイプ | Other | |||||
内容記述 | Article | |||||
ISSN | ||||||
収録物識別子タイプ | ISSN | |||||
収録物識別子 | 0099-2240 | |||||
書誌レコードID | ||||||
収録物識別子タイプ | NCID | |||||
収録物識別子 | AA00543249 | |||||
他の資源との関係:URI | ||||||
識別子タイプ | URI | |||||
関連識別子 | http://aem.asm.org/cgi/content/full/71/10/5692 | |||||
関連名称 | http://aem.asm.org/cgi/content/full/71/10/5692 | |||||
出版タイプ | ||||||
出版タイプ | AM | |||||
出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||
WoS | ||||||
表示名 | Web of Science | |||||
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