Molecular Mechanism on the Conjugation of Ciliate Blepharisma

1. blepharma japonicum中blepharone （gamone 1）偶联结构的研究是由互补交配型I和II相互作用诱导的，它们分别分泌blepharone （gamone 1）和blepharma moone （gamone 2）。Gamone 1转化II型细胞，使它们能够结合，而Gamone 2同样转化I型细胞。经鉴定，Gamone 1是一个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Con a亲和柱层析和天然PAGE纯化的新方法分离gamone 1。测定了从gamone 1中获得的4个多肽的部分氨基酸序列。利用来自交配型的mRNA，快速扩增分离到gamone 1 cDNA。该cDNA序列包含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4个n -乙酰氨基和3个甘露糖组成。为了阐明ble法利赛莫酮的受体，设计了ble法利赛莫酮的光亲和标记探针。虽然目前还没有成功的合成方法，但我们在此基础上合成了一个模型化合物n -苯甲酰- ble法利赛莫酮。模型化合物并不像预期的那样具有诱导交配的活性，而是具有显著的抑制交配的活性。

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.