Analysis of the codons for gene expression in rumen protozoa

本研究旨在阐明瘤胃原虫是否像自然环境中的四膜虫一样，使用TGA作为科尔终止密码子。方法上，从瘤胃原虫尾内甲藻cDNA文库中克隆基因。并得到以下结果。首先，我们成功地克隆了几丁质酶基因（ECchi 2）。对该基因进行分析，发现该基因ORF的终止密码子为TAG，与四膜虫的基因不同。TGA是四膜虫中唯一的终止密码子，位于3 '端poly（A）尾的下游，被认为与几丁质酶基因的表达无关。下一次获得的基因是微管蛋白基因（ECtub1）。在该ORF中，TAA通过通用密码子和位于其下游的TGA被识别为终止密码子，但在TAA之前存在识别谷氨酰胺（Q）的CAA，而且还有6个识别谷氨酰胺的CAA密码子。因此认为TAA是该ORF的终止密码子，并以几丁质酶为例，认为微管蛋白基因很可能使用识别TAA的通用密码子作为终止密码子。宫崎和UK的联合研究项目已经克隆了与微管蛋白一起构成细胞中微管的另一种成分的基因centrin基因，并揭示了该基因中的终止密码子是TAA。最终克隆的基因是酸性磷酸酶基因（ECaph1），该ORF的终止密码子是TAA。在该基因中，其中9个由CAA表达，1个由CAG表达，因此认为TAA是终止密码子。因此，认为瘤胃原虫尾内甲藻使用通用密码子。

This research was conducted to clarify whether rumen protozoa use TGA as a cole stop codon like Tetrahymena living in the natural habitat. Methodologically, genes cloned from cDNA library prepared from a rumen protozoon, Entodinium caudatum. were analyzed and following results were obtained. At first, we succeeded in cloning a chitinase gene (ECchi2). Then the gene was analyzed and the stop condon of the ORF in this gene was revealed to be TAG different from the genes of Tetrahymena. TGA known as a sole stop codon in Tetrahymena was found at the down stream of poly (A) tail in the 3'-terminal region and was thought not to relate with the expression of of the chitinase gene. The gene obtained next time was a tubulin gene (ECtub1). In this ORF.TAA was recognized as a stop codon through the universal codon and TGA located at the down streem of the TAA.However, there existed CAA which recognized glutamine (Q) just before the TAA and furthermore there were six more CAA codons for glutamine. Thus TAA was thought to be used as a stop codon in this ORF.It was also considered that the tubulin gene has a high possiblity to use universal codons recognizing TAA as a stop codon from the example of chitinase. A centrin gene which is a gene of another constituents of microtubule in the cells together with tubulin was already been cloned though the joint research project between Miyazaki and UK and it was revealed that a stop codon in this gene was TAA.The gene cloned finally was acid phosphatase gene (ECaph1) and a stop codon of this ORF was TAA.In this gene, ten glutamine was included and nine of them were expressed by CAA and another one by CAG.Thus TAA was thought to be used as stop codon. From all these results, rumen protozoon, Entodinium caudatum, was highly considered to use universal codons.