Molecular mechanisms of macronuclear differentiation during late stages of conjugation in the ciliated protozoan Tetrahymena

纤毛原生动物四膜虫接合后期大核分化的分子机制

• 批准号: 63540561
• 负责人: NUMATA Osamu
• 金额: $ 1.15万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63540561/
• 关键词: Ciliata; Tetrahymena; Intermediate filament protein; Citrate synthase; Macronuclear differentiation; Gene sharing; Micronuclear gene; Macronuclear gene; 小核; 大核; 遺伝子再構成; 接合

To investigate the molecular mechanisms of gene rearrangement during macro- nuclear differentiation, we have cloned and sequenced a Tetrahymena intermediate filament protein (49K protein) gene from a Tetrahymend thermophila macronuclear cDNA library using synthetic oligonucleotides as probes. The 49K protein showed an immunofluorescence localizations suggesting its involvement in the oral morphogenesis preceding binary fission and in nuclear events during fertilization, such as the selection of one of the four meiotic products, formation of the gametic pronuclei, gametic pronuclear transfer, and zygote formation. We have now found that the primary structure of 49K protein not only exhibits homology with those of mammalian vimentin and desmin but also shares 51.5% sequence identity with porcine heart citrate synthase. Furthermore, we demonstrate that 49K protein actually possesses citrate synthase activity, which indicates that 49K protein has dual functions as both a cytoskeletal protein and a respiratory enzyme. On the other hand, we succeeded in making a micronuclear genomic DNA library. Now we are cloning the 49K protein gene from the micronuclear genomic DNA library using 49K protein cDNA as probe.

为了研究巨核分化过程中基因重排的分子机制，我们利用人工合成的寡核苷酸作为探针，从嗜热四膜虫巨核cDNA文库中克隆并测序了一个四膜虫中间丝蛋白（49K蛋白）基因。49K蛋白的免疫荧光定位表明，它参与了口腔形态发生前的二元分裂和受精过程中的核事件，如四种减数分裂产物之一的选择、配子原核的形成、配子原核的转移和合子的形成。我们发现49K蛋白的一级结构不仅与哺乳动物的vimentin和desmin具有同源性，而且与猪心脏柠檬酸合成酶的序列同源性为51.5%。此外，我们证明49K蛋白实际上具有柠檬酸合成酶活性，这表明49K蛋白具有细胞骨架蛋白和呼吸酶的双重功能。另一方面，我们成功地建立了一个微核基因组DNA文库。目前，我们正以49K蛋白cDNA为探针，从微核基因组DNA文库中克隆49K蛋白基因。

项目成果

O.Numata, T.Takemasa, I.Takagi, M.Hirono, H.Hirano, J.Chiba, and Y.Watanabe: "Tetrahymena 14-nm filament-forming protein has citrate synthase activity" Nature.

O.Numata、T.Takemasa、I.Takagi、M.Hirono、H.Hirano、J.Chiba 和 Y.Watanabe：“四膜虫 14 nm 丝形成蛋白具有柠檬酸合酶活性”Nature。

広野雅文,沼田治,熊谷泰子,渡辺良雄: Proc.Natl.Acad.Sci.USA. 86. 75-79 (1989)

Masafumi Hirono、Osamu Numata、Yasuko Kumagai、Yoshio Watanabe：Proc.Natl.Acad.Sci.USA 86. 75-79 (1989)。

M.Hirono, Y.Kumagai, O.Numata, and Y.Watanabe: "Purification of Tetrahymena actin reveals some unusual properties" Proceedings of the National Academy of Sciences of the United States of America Vol.86, p.75-79, 1989.

M.Hirono、Y.Kumagai、O.Numata 和 Y.Watanabe：“四膜虫肌动蛋白的纯化揭示了一些不寻常的特性”美国国家科学院院刊第 86 卷，第 75-79 页，

I.Takagi,O.Numata,and Y.Watanabe.: "The roles of Tetrahymena 14-nm filament-forming protein and tubulin in dynamic behavior of gametic pronuclei at fertlization"

I.Takagi、O.Numata 和 Y.Watanabe.：“四膜虫 14 nm 丝形成蛋白和微管蛋白在受精时配子原核动态行为中的作用”

O.Numata,T.Takemasa,I.Takagi,M.Hirono,H.Hirono,J.Chiba,and Y.Watanabe.: "Tetrahymena 14-nm filament-forming protein has citrate synthase activity" Nature.

O.Numata、T.Takemasa、I.Takagi、M.Hirono、H.Hirono、J.Chiba 和 Y.Watanabe.：“四膜虫 14 nm 丝状形成蛋白具有柠檬酸合酶活性”Nature。