DNA sequencing of lysA gene of a rumen protozoan species

结合本课题负责人发现瘤胃原生动物具有从二氨基戊酸（DAP）合成赖氨酸的能力，本课题从常见的瘤胃原生动物Entodinium caudsturn的cDNA文库中筛选了一段用于生产赖氨酸（lysA）的DAPdecarboxylase编码基因，并测定了其dna序列，以明确该酶是否可以由瘤胃原生动物自身产生。首先，制备了在羊肚中只含尾尾绦虫作为原生动物的单羊。然后从山羊身上采集原生动物构建cDNA文库，利用需要赖氨酸的大肠杆菌突变体筛选赖氨酸。下一步，利用与6种细菌的lysA具有高度同源性的核苷酸序列制成探针，再次利用该文库用地高辛筛选lysA。结果发现了一个阳性斑块，并确定了更多的DNA序列。DNA序列分析结果显示，与草履虫的精尾特异性蛋白和表面抗原高度同源，而不是与lysA高度同源。再以6种细菌lysAs共有的同源性较高的核苷酸序列为引物，以cDNA文库为模板检测PCR产物，但未获得产物。由于如上所述每次试验都以失败告终，因此cDNA文库中似乎不存在lysA。最后，我们标记了尾藻细胞内的共生菌，并检测了DAP脱羧酶在细胞内的分布。结果表明，在8000和37000 x g离心后的沉淀具有较高的酶活性。通过透射电镜观察发现，两种沉淀中均含有大量的卵圆形细菌样颗粒，并且这些颗粒也存在于完整细胞的囊泡中。从颗粒中提取了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

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