Mechanism of Induction of Conjugation in Ciliates

本研究对纤毛虫，特别是异纤毛虫Blepharisma Japan的接合诱导的分子机制进行了研究。1.调节GAMONE-1基因在I型细胞中的表达，探讨了保证GAMONE-1表达的条件。我们发现，在适当的饥饿条件下，GAMONE 1基因在成熟的I型细胞中是限制性转录的。2.GAMONE 1基因在I型和II型细胞中的序列测定为了分析GAMONE 1基因在I型和II型细胞中的表达规律，我们克隆并测定了I型和II型细胞中GAMONE 1基因的基因组序列。研究表明，在I型和II型细胞中，基因组GAMONE 1基因中均不包含内含子。3.分析GAMONE-1基因编码接合诱导物质的基因表达规律，采用反向聚合酶链式反应技术，克隆并测定了I型细胞GAMONE-1基因上游868个碱基的序列。我们发现了两个TATA盒，一个CCAAT盒，一个PSE(近端序列元件)和几个已知转录因子识别的位点。4.在接合诱导细胞中特异表达的基因的鉴定。一些基因被认为在接合诱导的II型细胞中被激活。我们减去了处理细胞和未处理细胞之间的表达基因，并鉴定了几个在处理细胞中特异表达的基因。我们发现了编码CDK、CKS和4-HPPD的基因。这些基因在接合诱导后2-4小时内转录。

This study deals with the molecular mechanism of induction of conjugation in dliates, especially in heterotrich ciliate Blepharisma japonicum.1.Regulation of expression of gamone 1 gene in type I cells.We investigated the condition which ensures the gamone 1 expression. We revealed that gamone 1 gene is restrictively transcribed in mature type I cells under appropriate starved condition. The transcription was promoted by complementary gamone 2 and the level of starvation.2.Sequence determination of genomic gamone 1 gene in type I and type II cells.To analyze the regulation of gene expression of gamone 1 gene, we cloned and sequenced the genomic sequence of gamone 1 gene in type I and type II cells. The study revealed that intron was not included in the genomic gamone 1 gene in both type I and type II cells. The sequence of the gamone 1 gene in type n cells coincides with that of in type I cells.3.Analysis of the regulation of gene expression of gamone 1 gene encoding conjugation-inducing substance.Using inverse PCR technique, we cloned and sequenced 868 bp of the upstream of gamone 1 gene in type I cells. We found two TATA boxes, a CCAAT box, a PSE (proximal sequence element) and several sites which are recognized by known transcriptional factors.4.Identification of genes which are specifically expressed in conjugation-induced cells.Several genes are considered to be activated in conjugation-induced type II cells. We subtracted expressed genes between gamone-treated and untreated cells, and identified several genes which were specifically expressed in gamone-treated cells. We found genes encoding Cdk, Cks and 4-HPPD. These genes were transcribed in 2-4 hours after induction of conjugation.