Structural and Functional Analysis of Linear Plasmid DNAs of Plant Pathogenic Fungus

1.在114个立枯丝核菌田间分离物中，有48个分离物含有质粒样DNA。这48个分离物在日本分离物的9个吻合群（AG）和种内群（ISG）中的分布如下：solani：AG-2-1、AG-2 - 2、AG-3、AG-4、AG-5、AG-6分别为0、1、0、11、10、11、11每个分离物携带一个、两个或三个通过凝胶电泳鉴定的plDNA。电子显微镜分析表明，所有这些plDNA都是线性分子。通过Southern印迹分析，使用切口翻译的plDNA作为探针，检查在代表性分离株中发现的plDNA之间的序列同源性。在从相同AG和ISG内的分离物获得的pIDNA之间观察到相当大的序列同源性。根据序列同源性将AG-2-2菌株中存在的plDNAs分为两类.在立枯丝核菌融合群4（AG-4）的RI-64菌株中发现了3个线性DNA质粒。这些质粒命名为pRS 64 -1、pRS 64 - 2和pRS 64 - 3，具有相同的2.7kb大小。对pRS 64 -1、-2和-3的限制性酶切图谱和Southern杂交分析显示在两个末端存在同源区域。即使用蛋白酶K或碱处理后，质粒DNA也对3 '-外切核酸酶和5'-外切核酸酶具有抗性。通过限制性内切酶消化产生的两个末端片段的长度在变性条件下加倍，表明线性质粒DNA在两个末端都具有发夹环。总DNA的Southern印迹分析显示存在两种类型的pRS 64 DNA的二聚体形式。一种是头对头二聚体，另一种是尾对尾二聚体。这些独特的DNA结构在细胞复制中的作用

1. Plasmid-like DNA (plDNA) was found in 48 out of 114 field isolates of Rhizoctonia solani. These 48 isolates were distributed as follows among the nine anastomosis groups (AG) and intraspecific groups (ISG) recognized among Japanese isolates of R. solani : 0 in AG-1 (sasakii type), 1 in AG-1 (web-blight type), 0 in AG-2-1, 11 in AG-2-2 (rush type), 10 in AG-2-2 (root rot type), 10 in AG-3, 8 in AG-4, 4 in AG-5 and 4 in AG-6. Each isolate carried one, two or three plDNAs identified by gel electrophoresis. Electron microscopic analysis revealed that all these plDNAs were linear molecules. The sequence homology among plDNAs found in representative isolates was examined by Southern blot analysis, using nick-translated plDANs as probes. Considerable sequence homology was observed among plDNAs obtained from isolates within the same AG and ISG. The plDNAs occurring in the isolates of AG-2-2 were classified into two groups on the basis of the sequence homology.2. Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization anlysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with protease K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of th