权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of transfer genes in plasmid R64

质粒 R64 中转移基因的分析

基本信息

批准号：
06454005
负责人：
KOMANO Teruya
金额：
$ 4.48万
依托单位：
TOKYO METROPOLITAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454005/
关键词：
Plasmid R64 Conjugation Shufflon Thin pilus

项目摘要

Conjugal transfer is an important process to exchange genetic material among bacterial cells. Forty-nine genes were found in the 54-kb transfer region of plasmid R64. To test the requirement of these genes for R64 conjugation, deletion and insertion mutations were introduced into all genes. For R64 surface mating, 24 transfer genes were found to be essential, while for liquid mating, additional 12 genes were required. The pnd gene was responsible for plasmid stability. Many gene products were detected by the maxicell method. The 12 genes required for liquid mating were responsible for the formation of R64 thin pilus. The purified R64 thin pilus consists of the pils and pilV products. The pils product was first synthesized as 22-kDa protein, and then processed to 19-kDa mature pilin mediated by the pilU product. To reveal the relationship between structure and function of the pilS gene, many pils mutants were isolated. DNA rearrangement of the shufflon selects one of the seven pilV genes with different C-terminal segments and determined the recipient specificity in liquid mating. Lipopolysaccharides in the recipient cells are suggested to function as receptors for pilV proteins from mating experiments using various rfa mutants of E.coli k-12 and S.typhimurium LT2 as recipient strains. The rci gene encoding shufflon-specific recombinase was overxpressed and the Rci protein was purified. An in vitro recombination system was constructed using the purified Rci protein. A plasmid cyrrying two tandemly repeated oriT sequences was constructed. A recombination event to lose a DNA segment between two oriT sequences was observed after mobilization. A recombination depending nikAB genes was also observed in the donor cells.

配偶转移是细菌细胞间交换遗传物质的重要过程。在R64的54kb转移区发现了49个基因。为了测试这些基因对R64接合的要求，在所有基因中引入了缺失和插入突变。对于R64的表面交配，24个转移基因是必需的，而对于液体交配，则需要另外12个基因。PND基因是决定质粒稳定性的关键基因。用Maxicell方法检测到多种基因产物。液体交配所需的12个基因负责R64细菌毛的形成。纯化的R64薄菌毛由Pils和PilV产品组成。Pils产物首先被合成为22 kDa的蛋白质，然后在Pilu产物的介导下加工成19 kDa的成熟Pilin。为了揭示PILS基因的结构和功能之间的关系，我们分离到了许多PIRS突变体。洗牌子的DNA重排选择了7个C末端片段不同的PilV基因中的一个，并决定了液体交配中的受体特异性。受体细胞中的脂多糖被认为是PilV蛋白的受体，通过以E.coliK-12和鼠伤寒沙门氏菌LT2的不同RFA突变体作为受体菌株的交配实验。表达了重组酶基因RCI，并纯化了RCI蛋白。用纯化的RCI蛋白构建了体外重组系统。构建了含有两个重复的ORIT序列的重组表达载体。动员后观察到两个ORIT序列之间的DNA片段丢失的重组事件。在供体细胞中也观察到依赖于nikab基因的重组。