DNA sequencing of lysA gene of a rumen protozoan species

鉴于首席调查员已发现瘤胃原生动物具有从二氨基二胺(DAP)合成赖氨酸(Lysa)的能力，本课题从一种常见的Ruinen原生动物Etodina caudsturn的cDNA文库中筛选编码DAP脱羧酶以产生赖氨酸(Lysa)的基因，并测定其DNA序列，以阐明该酶是否可由原生动物自身产生。首先，在林嫩地区制备了只含有尾型棘球绦虫作为原虫的单群山羊。然后，从山羊采集的原生动物中构建了cDNA文库，并利用大肠杆菌赖氨酸需求突变株对Lysa进行筛选。然而，结果是阴性的，下一步，利用与六种细菌lysA高度同源的核苷酸序列制成的探针，再次利用该文库筛选含有地高辛配基的Lysa。结果发现一个阳性斑块，其dna序列为…更多的NCE被确定下来。然而，DNA序列分析的结果表明，它与草履虫的精子尾部特异蛋白和表面抗原有很高的同源性，而不是Lysa。以该文库为模板，以6种细菌的DNA序列同源性较高的核苷酸序列为模板，对扩增产物进行检测，均未得到扩增产物。由于如上所述，每一次试验都以失败告终，Lysa似乎不存在于cDNA文库中。最后，我们在尾巴乳杆菌的细胞中标记了内共生菌，并检测了细胞内DAP脱羧酶活性的分布。结果表明，经8000和37000×g离心后的沉淀物具有较高的酶活力。透射电子显微镜观察表明，两种沉淀物中均含有大量椭圆形细菌样颗粒，且存在于完整细胞的囊泡中。已经从这些颗粒中提取了DNA(20,000个碱基)。我们已经开始从DNA中再次筛选Lysa，并期望达到最初的目标。较少

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