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Analyses of Clustered Inversion Region in Incl Plasmids.

包含质粒中成簇反转区域的分析。

基本信息

批准号：
01540533
负责人：
KOMANO Teruya
金额：
$ 1.15万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01540533/
关键词：
Plasmid Clustered inversion region Shufflon DNA rearrangement Conjugal transfer

项目摘要

Shufflon, or clustered inversion region, is a novel type of DNA rearrangement found in the IncI1 plasmid R64. The shufflon of R64 consists of four DNA segments, designated A, B, C and D, which differ in DNA length and sequence. The four DNA segments are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of the seven C-termini of a gene, pilV.By constructing a series of deletion-derivatives of plasmid R64, a region responsible for the conjugal transfer was located within a 54-kb DNA region to the right of replication region. We determined the nucleotide sequence of a 19-kb DNA region responsible for the formation of thin pilus, and found that 18 open reading frames (ORFs) are present in the same orientation. The pliv gene is the last one of the 18 ORFs. By introducing deletion and inser … More tion mutations into all the 18 ORFs, we demonstrated that most of the 18 ORFs are required for the formation of thin pilus and for the conjugal transfer. To reveal the role of the DNA rearrangement in the shufflon region, we constructed R64 derivatives where the C-termini of the pilv gene are fixed. Donor cells carrying R64 derivative plasmids with the seven fixed pilv C-termini were mated with one of several recipient bacterial strains including E. Coli K-12, B and C, Salmonella typhimurium LT2 and Shigella flexneri. The results indicated that the transfer frequencies of liquid mating dramatically change according to the combination of recipient strains and variable C-terminiof the pilv gene in the donors, suggesting that the shufflon determines the recipient specificity in liquid mating in plasmid R64. We also sequenced the oriT region located at the other end of transfer region of R64 and found two open reading frames, designated nikA and nikB. NikA and nikB were found indispensable for the transfer of R64 and were demonstrated to function together as an oriT-specific nickase. Less

Shufflon或成簇倒位区是在IncI 1质粒R64中发现的一种新型DNA重排。R64的改组由四个DNA片段组成，命名为A、B、C和D，它们在DNA长度和序列上不同。这四个DNA片段的侧翼由七个19-bp的重复序列隔开。任何两个反向重复序列之间的位点特异性重组导致复杂的DNA重排。开放阅读框分析表明，shufflon是选择pilV基因7个C-末端之一的生物开关。通过构建一系列质粒R64的缺失衍生物，将负责接合转移的区域定位在复制区右侧54-kb的DNA区域内。我们确定了负责薄菌毛形成的19-kb DNA区域的核苷酸序列，并发现18个开放阅读框（ORF）以相同的方向存在。pliv基因是18个ORF中的最后一个。通过引入删除和插入 ...更多信息通过对所有18个ORF的突变，我们证明了18个ORF中的大多数是薄菌毛形成和接合转移所必需的。为了揭示DNA重排在shufflon区域中的作用，我们构建了R64衍生物，其中pilv基因的C-末端被固定。将携带有7个固定菌毛C末端的R64衍生质粒的供体细胞与包括大肠杆菌在内的几种受体细菌菌株之一交配。大肠杆菌K-12、B和C、鼠伤寒沙门氏菌LT 2和福氏志贺菌。结果表明，液体交配的转移频率随受体菌株和供体pilv基因C末端的不同而发生显著变化，表明重组子决定了质粒R64液体交配的受体特异性。我们还对位于R64转移区另一端的oriT区进行了测序，发现了两个开放阅读框，命名为nikA和nikB。发现NikA和nikB对于R64的转移是必不可少的，并且被证明作为oriT特异性切口酶一起起作用。少