Random insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes
Random insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes
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DOI：
10.1111/j.1574-6968.2001.tb10955.x
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发表时间：
2001-12-18
影响因子：
2.1
通讯作者：
Meijer, WG
中科院分区：
文献类型：
--
作者：
Mangan, MW;Meijer, WG
由于缺乏体内随机插入突变的方法，严重阻碍了对马红球菌毒力因子的鉴定。本研究利用转座体将卡那霉素抗性基因随机插入到马立克氏酵母33701的基因组中。以卡那霉素抗性基因为探针的Southern杂交结果表明，转座体的插入是随机的。在对转座体和染色体DNA之间的连接进行核苷酸序列分析后，证实了这一点。靶序列9bp重复的存在表明转座体的随机整合是由于真实的Tn5转座事件。(C)2001年欧洲微生物学会联合会。爱思唯尔科学公司出版。版权所有。
The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis. This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R. equi ATCC 33701. Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random. This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA. The presence of a 9 bp duplication of the target sequence showed that-random integration of the transposome was due to a bona fide Tn5 transposition event. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.