Analysis of the gene of lysine-synthesizing enzyme in rumen protozoa

瘤胃原生动物的基因分析尚未深入研究。在目前的研究中出现了一些意想不到的问题，耽误了我们研究计划的完成。然而，本研究在基因分析的研究领域取得了一定的成果，在国际上首次确定了尾尾肠赖氨酸合成酶（lysA: diaminopimelate decarboxylase）的部分基因。该结果为进一步开展瘤胃原生动物基因分析提供了基础数据。我们在这项研究开始时遇到的第一个问题是用于纯化mRNA的原生动物的起源。从一种非共生培养物中衍生出来的原生动物给了我们太少的mRNA来制造cDNA。从只接种尾尾绦虫的单一驯化山羊身上收集的原生动物给我们提供了足够的mRNA。建立cDNA文库后，首先用大肠杆菌赖氨酸需要突变体筛选lysA基因。但该方法因细菌对溶素缺乏的反应消失而中断。这是一个悬而未决的问题。然后，考虑到一般原生动物（毛虫）的密码子使用（特别是停止密码子）与通用密码子不同，利用序列为5‘TTAAATCTTGGAGGCGGATTC3’的探针从cDNA文库中筛选原生动物的lysA基因，该基因与几种细菌的lysA基因具有高度同源性，并标记为地高高金，检测到阳性斑块。克隆其中一个空斑，用链终止法确定插入片段的DNA序列。尾尾原虫与其他纤毛虫原生动物一样使用TAA作为终止密码子，与其他纤毛虫一样使用TGC识别半胱氨酸，与已知使用TGA识别半胱氨酸的原虫不同。

Gene analysis of rumen protozoa has not been explored. The happening of some unexpeted problems in the present study has delayd the accomplishment of our research plan. As a result, however, the present study reached for sure at the research field of gene analysis and a part of the gene of lysine-synthesizing enzyme (lysA : diaminopimelate decarboxylase) of Entodinium caudatum has been determined for the first time in the world. The results seemed meaningful as fundamental data for continuing the gene analysis of the rumen protozoa in future. The first problem we met at the beggining of this study was the origin of the protozoon to be used for purifying mRNA.The protozoon derived from an agnotobiotic culture gave us too small amount of mRNA to make cDNA.The protozoon collected from a monofaunated goat, to which only E.caudatum had been inoculated, gave us an enough amount of mRNA.After making cDNA library, screening of lysA gene was carried out at first with lysine-requiring mutant of E.coli. But the method was interrupted by disappearance of the response of the bacterium to lysin-deficiency. This was left as a pending issue. Then, considering the fact that the codon usage (especially, stop codon) of protozoa (ciliates) in general is different from the universal one, lysA gene of the protozoon was screened from the cDNA library using a probe with a sequence 5'TTAAATCTTGGAGGCGGATTC3', which had a high homology in lysA gene of several bacteria and labelled with digoxigenin, and positive plaques were detected. One of these plaques was cloned and the DNA sequence of the insert was determined by the chain termination method. The protozoon, E.caudatum, seemed to use TAA as a stop codon like Euplotes (one of ciliates) and different from other ciliated protozoa, and to use TGC for recognizing cysteine like other organisms and different from Euplotes which had known to use TGA for cysteine.