Mechanism of Induction of Conjugation in Ciliates

本研究探讨了纤毛虫，特别是异毛纤毛虫Blepharisma japonicum接合诱导的分子机制。1.染色体1基因在I型细胞中表达的调节。我们研究了确保染色体1表达的条件。我们发现，gamone 1基因是限制性转录在成熟的I型细胞在适当的饥饿条件下。2. Ⅰ型和Ⅱ型细胞基因组中gamone 1基因的序列测定为了分析gamone 1基因的表达调控，我们对Ⅰ型和Ⅱ型细胞中gamone 1基因的基因组序列进行了克隆和测序。研究表明，内含子不包括在基因组配子1基因在I型和II型细胞。3.分析结合诱导物基因的表达调控：利用反向PCR技术，克隆并测序了I型细胞中gamone 1基因上游868 bp的序列。我们发现了两个TATA盒、一个CCAAT盒、一个PSE（proximal sequence element）和几个已知转录因子识别的位点。4.在接合诱导的细胞中特异表达的基因的鉴定我们减去了配子处理和未处理的细胞之间的表达基因，并确定了几个基因，这些基因在配子处理的细胞中特异性表达。我们发现了编码Cdk、Cks和4-HPPD的基因。这些基因在诱导接合后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.