八肋游仆虫第二类释放因子基因的克隆与序列分析

Cloning and Sequence of eRF3 Gene from Euplotes octocarinatus

分离八肋游仆虫 (Euplotesoctocarinatus)大核eRF3基因 ,为进一步研究第二类释放因子结构与功能 ,探讨低等真核生物新生肽链释放机理提供实验素材 .以八肋游仆虫基因组DNA为材料 ,根据已知的第二类释放因子eRF3保守氨基酸序列设计引物 ,扩增克隆了该游仆虫的第二类释放因子基因片段 ,并对其核苷酸序列进行了分析 .根据测得的序列设计特异性引物 ,并利用游仆虫的端粒序列 (C4 A4 C4 A4 C4 A4 C4 )为引物 ,扩增得到该基因的全序列 .序列分析表明 ,该基因位于 2 782bp长的大核染色体上 ,编码区由 2 4 0 0bp组成 ,编码 80 0个氨基酸 。

The aim was to isolate the release factor 3(eRF3) gene from the macronuclear DNA of Euplotes octocarinatus for further study of the structure and function of eRF3 and to understand the mechanism of termination of translation in lower eukaryotes. According to the reported amino acid sequence of eRF3, two generated primers were designed and synthesized. The eRF 3 gene fragment was amplified from the genomic DNA of \%Euplotes octocarinatus\%. The fragment was cloned and sequenced. Gene specific primers were then synthesized. Using Euplotes telomeric sequence (C\-4A\-4C\-4A\-4C\-4A\-4C\-4) as primer in combination with the gene specific primer, additional eRF 3 fragments were amplified and cloned. From these overlapping fragments the complete gene sequences coding for the eRF3 of \%E. octocarinatus\% was obtained. Analyzing of the nucleotide sequence show that the macronuclear chromosome, containing the whole gene of eRF3 is 2 787 bp long. It contains an open reading frame of 2 400 bp and encodes 800 amino acids.Further characteristics of the gene and the deduced amino acid sequence were discussed.