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Molecular Mechanism on the Conjugation of Ciliate Blepharisma

纤毛虫眼睑接合的分子机制

基本信息

批准号：
11680592
负责人：
IIO Hideo
金额：
$ 2.3万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

1. Studies on the structure of blepharmone (gamone 1)Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Gamone 1 was isolated and characterized as a glycoprotein of 20-30 kDa containing 175 amino acids and 6 sugars. To establish the structure of gamone 1, it was isolated by a new method based on concentration by Con A affinity column chromatography and purification by native PAGE. The partial amino acid sequence of four peptides obtained from gamone 1 was determined. By using mRNA from mating type, gamone 1 cDNA was isolated by rapid amplification. The cDNA sequence contains an ORF of 305 amino acids and codes a novel protein. We also estimated that the sugar sequence for the glycoprotein consisted from four N-acetylglucosamines and three mannoses.2. Studies on the receptor of blepharismone (gamone 2)To elucidate the receptor of blepharismone, a photoaffinity label probe of blepharismone is designed. Although the synthesis of it has not been attained so far, we synthesized a model compound N-benzoyl-blepharismone based on the strategy developed before. The model compound did not have mating inducing activity as expected, it was found to haye the significant activity of mating inhibition.

1. Blepharisma japonicum中的接合是由互补交配型I和II之间的相互作用诱导的，它们分别分泌Blepharmonone（gamone 1）和Blepharismone（gamone 2）。配子1转化II型细胞，使它们能够结合，配子2类似地转化I型细胞。Gamone 1被分离并表征为20-30 kDa的糖蛋白，含有175个氨基酸和6个糖。为了确定gamone 1的结构，通过基于ConA亲和柱层析浓缩和非变性PAGE纯化的新方法分离gamone 1。测定了从配子1中获得的四个肽的部分氨基酸序列。以交配型的mRNA为模板，通过快速扩增法分离配子体1的cDNA。该cDNA序列含有305个氨基酸的ORF，编码一种新的蛋白质。我们还估计糖蛋白的糖序列由四个N-乙酰葡糖胺和三个甘露糖组成。为了研究小泡霉素受体，设计了一种小泡霉素的光亲和标记探针。虽然它的合成至今还没有实现，我们合成了一个模型化合物N-苯甲酰基-blepharismone的基础上开发的策略。该模型化合物没有预期的交配诱导活性，但具有明显的交配抑制活性。