Molecular Mechanism on the Conjugation of Ciliate Blepharisma

1.日本血吸虫(Blepharisma Japan Onicum)白花素(Gamone 1)结合结构的研究是由互补交配型I型和II型交配诱导的，它们分别分泌Blepharisma Japan(GAMONE 1)和BEFERARISMone(GAMONE 2)。Gamone 1转化II型细胞，使它们能够结合，Gamone 2类似地转化I型细胞。Gamone 1是一种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，采用ConA亲和柱层析浓缩和天然PAGE纯化的新方法对其进行分离纯化。测定了从GAMONE 1中分离得到的4个多肽的部分氨基酸序列。以交配型mRNA为模板，通过快速扩增的方法获得了GAMONE-1基因。该序列包含305个氨基酸的开放阅读框架，编码一个新的蛋白质。我们还估计了该糖蛋白的糖序列由四个N-乙酰氨基葡萄糖和三个甘露糖组成。眼睑松受体(甘丙酮2)的研究为了阐明眼睑松受体，设计了一种眼睑松光亲和标记探针。虽然到目前为止它的合成还没有实现，但我们在此基础上合成了一种模型化合物N-苯甲酰-眼睑松酮。模型化合物没有达到预期的交配诱导活性，有显著的交配抑制活性。

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.