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Analysis of the codons for gene expression in rumen protozoa

瘤胃原虫基因表达密码子分析

基本信息

批准号：
09460130
负责人：
ONODERA Ryoji
金额：
$ 8.45万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

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.

本研究旨在澄清瘤胃原生动物是否像生活在自然栖息地的四膜虫一样使用 TGA 作为油菜终止密码子。方法学上，从瘤胃原生动物 Entodinium caudatum 制备的 cDNA 文库中克隆基因。进行了分析并得到以下结果。首先，我们成功克隆了几丁质酶基因（ECchi2）。对该基因进行分析，发现该基因ORF的终止密码子是与四膜虫基因不同的TAG。 TGA是四膜虫中唯一的终止密码子，存在于poly(A)尾3'末端区域的下游，被认为与几丁质酶基因的表达无关。下次获得的基因是微管蛋白基因(ECtub1)。在该ORF中，TAA通过位于TAA下游的通用密码子和TGA被识别为终止密码子。然而，在TAA之前存在识别谷氨酰胺(Q)的CAA，此外还有另外6个识别谷氨酰胺的CAA密码子。因此，TAA被认为用作该ORF中的终止密码子。从几丁质酶的例子中，还认为微管蛋白基因很有可能使用识别TAA的通用密码子作为终止密码子。宫崎骏和英国的联合研究项目已经克隆了与微管蛋白一起构成细胞内微管的另一种成分的基因centrin基因，并发现该基因的终止密码子是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。该基因中包含10个谷氨酰胺和9个谷氨酰胺。它们由CAA表达，另一种由CAG表达。因此TAA被认为被用作终止密码子。从所有这些结果来看，瘤胃原生动物 Entodinium caudatum 被高度认为使用通用密码子。