MOLECULAR CELL BIOLOGICAL STUDIES ON INTRACELLULAR SYMBIOSIS

本研究对Buchnera、蚜虫胞内共生菌和褐飞虱胞内类酵母共生菌进行了研究，主要结果如下：1。比较幼龄和老龄蚜虫的菌细胞共生系统的基因表达模式发现，只有当共生系统保持功能时，宿主细胞和Buchnera的基因才特异性表达。这些基因包括那些参与氨基酸代谢的酶，证实了我们以前的结果。获得了Buchnera产生核黄素并为宿主提供维生素的有力证据.我们估计的基因组大小Buchnera的基因组DNA与限制性内切酶的限制性位点是丰富的G/C，然后通过脉冲场凝胶电泳。结果发现，基因组大小为657 kbp，仅为大肠杆菌基因组大小的七分之一。两个不同基因的斑点印迹分析和细胞DNA含量的直接荧光测定法显示，Buchnera是非常特别的细菌，在单个细胞中具有超过100个基因组拷贝。此外，Buchnera的基因组拷贝数在很大程度上是可变的，这是由于宿主昆虫的形态和发育阶段。Buchnera作为细菌是例外的，因为它们含有组蛋白Hi样蛋白，其可能与基因组DNA相互作用。我们成功地克隆了酵母样共生体的尿酸酶基因，该基因是稻飞虱动员储存在组织中的尿酸所必需的。此外，我们通过将尿酸酶基因连接到表达载体中转化大肠杆菌，构建了尿酸酶基因的大规模表达系统。

We studied on Buchnera, intracellular symbiotic bacteria of aphids and intracellular yeast-like symbionts of brown planthoppers, the results of which are summarized as follows :1. Comparison of gene expression patterns of the bacteriocyte symbiotic systems from young and old aphids revealed that there are more than ten genes, of both host cell and Buchnera, that are specifically expressed only when the symbiotic system is kept functional. These genes include those for enzymes involved in the amino acid metabolism, confirming our previous results.2. We attained convincing evidence that Buchnera produce riboflavin and provision the vitamin to hosts.3. We estimated the genome size of Buchnera by digesting the genomic DNA with restriction enzymes whose restriction sites are rich in G/C, followed by pulse-field gel electrophoresis. It turned out that the genome size, 657kbp, is only one seventh of that of the genome of E.coli.4. Dot-blot analyses of two different genes and direct fluorimetry of the cellular DNA content revealed that Buchnera are very extraordinary bacteria with more than 100 genomic copies in a single cell. In addition, the genomic copy number of Buchnera is variable to a great extent due to morph and developmental stage of the host insect.5. Buchnera are exceptional as bacteria in that they contain a histone Hi -like protein, which is likely in interaction with the genomic DNA.6. We successfully cloned the gene for uricase of the yeast-like symbiont that is essential for planthoppers to mobilize the uric acid stored in their tissue. Also, we constructed a large scale expression system of the uricase gene by transforming E.coil with the gene ligated into an expression vector.