Studies on the origin of eukaryotes-Functional analyzes of ribosomes of microsporidia

真核生物起源研究——小孢子虫核糖体功能分析

• 批准号: 07640936
• 负责人: INOUE Tadashi
• 金额: $ 0.96万
• 依托单位: The Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640936/
• 关键词: Nosema bombycis; Ribosome; in vitro protein synthesis; 微胞子虫; 試験管内蛋白質合成系; リボソーム; 真核生物

In order to investigate the evolutionary origin of eukaryotes, we attempted to develop an in vitro protein synthesizing system consisting of ribosomes from some microsporidia including Nosema bombycis which is believed to diverge at early stage of eukaryotes. N.bombycis is known to have ribosomes whose sedimentation property is very similar to that of prokaryotes.Crude cellular extract was prepared from both dormant and artificially hatched spores. Ribosomes was recovered from the extract by differential centrifugation and incubated with the soluble enzyme fraction in an in vitro protein synthesizing system. The system contained exogenously added poly (U), ^3H-Phe and an ATP-regenarating system. The amount of in vitro protein synthesis was measured by counting the radioactivity of hot trichloroacetic acid-insoluble fraction after incubation.All the attempt to establish the system was failed because of poor activity of ribosomes and/or enzyme fraction of N.bombycis. Supplementation of the enzyme fraction of Ecsherichia coli, rat liver and midgut of silkworm had no effect on the activity. Aminoacyl-tRNA synthesis, however, took place normally in the system from N.bombycis. These results may indicate that some inhibitory factors are present in the preparation of N.bombycis ribosomes.

为了研究真核生物的进化起源，我们尝试建立了一个由包括家蚕微孢子虫在内的一些微孢子虫核糖体组成的体外蛋白质合成系统，该系统被认为在真核生物的早期阶段分化。家蚕新孢子具有与原核生物相似的核糖体沉降特性，从休眠孢子和人工孵化的孢子中提取细胞粗提物。通过差速离心从提取物中回收核糖体，并在体外蛋白质合成系统中与可溶性酶级分孵育。该系统含有外源性添加的聚（U）、^3 H-Phe和ATP再生系统。体外蛋白质合成量的测定是通过计算孵育后热三氯乙酸不溶部分的放射性来进行的，但由于家蚕核糖体和/或酶部分活性差，所有建立该系统的尝试都失败了。添加大肠杆菌、大鼠肝和家蚕中肠酶组分对酶活性无影响。氨酰-tRNA的合成，然而，发生正常的系统从N. bombycis。这些结果可能表明，在家蚕核糖体的制备过程中存在一些抑制因子。

项目成果

Yanagisawa, H.: "A unique origin and multistep process for generation of expanded DRPLA tripet repeats." Hum. Mol. Genet.5. 373-380 (1996)

Yanagisawa, H.：“用于生成扩展 DRPLA 三联体重复序列的独特起源和多步骤过程。”

Kawakami, Y., Inoue, T., Uchida, Y., Hatakeyama, Y., Iwano, H.and Ishihara, R.: "Specific amplification of DNA from reference strains of Nosema bombycis (Nosematidae, Microsporidia)" J.Seric.Sci.Jpn.64. 165-172 (1995)

Kawakami, Y.、Inoue, T.、Uchida, Y.、Hatakeyama, Y.、Iwano, H. 和 Ishihara, R.：“家蚕微孢子虫（微孢子虫科、微孢子虫）参考菌株 DNA 的特异性扩增” J.Seric

Mizuno, T.: "Identification of the nuclear localization signal of mouse DNA primase: Nuclear transport of p46 subunit is facilitated by integration with p54 subunit." J. Biol. Chem.(in press). (1996)

Mizuno, T.：“小鼠 DNA 引物酶核定位信号的鉴定：p46 亚基的核转运通过与 p54 亚基的整合而促进。”