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DNA sequencing of lysA gene of a rumen protozoan species

瘤胃原生动物 lysA 基因的 DNA 测序

基本信息

批准号：
08044215
负责人：
ONODERA Ryoji
金额：
$ 4.1万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044215/
关键词：
Rumen protozoa Entodinium caudatum cDNA library Lysine-requiring mutant of Escherichia coli lysA screening Diamionopimelate Transmission electron micrograph Endosymbiont エンドシンピオントジゴキシゲニン Entodinium caudatuin ジアミノピメリン酸脱炭酸酵素遺伝子 LysA

项目摘要

In connection with the fact that the head investigator had discovered an ability of the rumen protozoa to synthesize lysine from diaminopirnelate (DAP), this project was conducted to screen a gene for coding DAPdecarboxylase to produce lysine (lysA) from a cDNA library of Entodinium caudsturn, one of the popular ruinen protozoa, and to determine its DNAsequence, in order to clarify whether or not the enzyme could be produced by the protozoa themselves. At first, mono-faunated goats which contain only E.caudatum as protozoa in the rinnen were prepared. Then the cDNA library was made from the protozoa collected from the goats and was used for screening lysA using lysine-requiring mutant of Escherichia coli. However, the result was negative, In the next step, the library was again used for screening lysA with digoxigenin using a probe made from the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria. As a result a positive plaque was found and its DNA seque … More nce was determined. The result of the analysis of the DNA sequence, however, revealed high homologies not with lysA but with sperm-tail specific proteins and surface antigen of Paramecium. Then the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria was again used as primers to examine PCR products using the cDNA library as a template, but no products were obtained. Since every trial resulted in failure as mentioned above, lysA seemed not to exist in the cDNA library. Finally we marked endosymbionts in the cells of E.caudatwn and examined the distribution of DAP decarboxylase activity in the cells. it was shown that the precipitates after centrifugation at 8,000 and 37,000 x g had high activity of the enzyme. Observations of the precipitates by a transmission electron microscope revealed that a lot of oval bacterium-like particles were included in both precipitates and the particles also existed in vesicles in the intact cells. DNA (20,000 bp) has been extracted from the particles. We already started screening lysA again from the DNA and expect to attain the initial objective. Less

本课题结合研究者发现瘤胃原生动物具有从二氨基嘌呤酸（DAP）合成赖氨酸的能力，从尾内甲藻（Entodinium caudsturn）的cDNA文库中筛选编码DAP脱羧酶产生赖氨酸（lysA）的基因，并测定其DNA序列，以澄清这种酶是否可以由原生动物自身产生。首先，制备了在林嫩中仅含有尾状艾美耳球虫作为原生动物的单一动物群山羊。然后从山羊体内采集原虫，构建cDNA文库，并利用大肠杆菌的赖氨酸需要突变体筛选lysA。然而，结果是阴性的。在下一步中，使用由与六种细菌的lysA共有的具有高度同源性的核苷酸序列制成的探针，再次将文库用于用地高辛筛选lysA。结果发现一个阳性空斑，其DNA序列与正常对照组比较，差异有统计学意义（P < 0.05）。 ...更多信息 nce是确定的。DNA序列分析结果表明，该基因与草履虫精子尾部特异蛋白和表面抗原的同源性较高，而与lysA的同源性较低。然后，再次使用与六种细菌的lysA具有高度同源性的核苷酸序列作为引物，以cDNA文库为模板检测PCR产物，但没有获得产物。如上所述，由于每次试验都失败，因此cDNA文库中似乎不存在lysA。最后，我们标记了尾状艾美耳球虫细胞内的内共生菌，并检测了DAP脱羧酶活性在细胞内的分布。结果表明，在8，000和37，000 × g下离心后的沉淀物具有高的酶活性。用透射电镜观察沉淀物，发现两种沉淀物中都含有大量的椭圆形细菌样颗粒，完整细胞中的囊泡中也含有这些颗粒。从颗粒中提取DNA（20，000 bp）。我们已经开始从DNA中再次筛选lysA，并期望达到最初的目标。少