OMEGON-KM - A TRANSPOSABLE ELEMENT DESIGNED FOR INVIVO INSERTIONAL MUTAGENESIS AND CLONING OF GENES IN GRAM-NEGATIVE BACTERIA
OMEGON-KM - A TRANSPOSABLE ELEMENT DESIGNED FOR INVIVO INSERTIONAL MUTAGENESIS AND CLONING OF GENES IN GRAM-NEGATIVE BACTERIA
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DOI：
10.1016/0378-1119(89)90162-5
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发表时间：
1989-03-30
期刊：
影响因子：
3.5
通讯作者：
FREY, J
中科院分区：
文献类型：
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作者：
FELLAY, R;KRISCH, HM;FREY, J
为了结合Omega插入子的特点和体内转座突变的优点，我们构建了一个人工转座子，称为Omegon-KM。Omegon-KM转座子携带在pJFF350上，可以接合动员到广泛的革兰氏阴性菌中。Omegon-Km的两侧是从IS1末端衍生的合成的28个碱基重复序列，方向相反。此外，Omegon-KM的每一端都有非常有效的OMEGA插入子的转录和翻译终止子。在体内，Omegon-KM携带可选择的卡那霉素(KM)-新霉素抗性基因(APHA)，该基因在许多革兰氏阴性菌中都有很好的表达。IS1转座功能位于供体质粒上，但位于Omegon-KM的外部。因此，Omegon-KM的插入非常稳定，因为它们缺乏进一步换位的能力。Omegon-KM突变是通过将pJFF350从大肠杆菌接合转移到任何该质粒不能复制的革兰氏阴性受体菌株中进行的。那些已经发生转座事件的细胞是通过它们对Km的抗性来选择的。恶臭假单胞菌的Omegon-KM转座频率很高。对其他革兰氏阴性土壤和水细菌(豆科根瘤菌、脱氮副孢子菌)的初步试验产生了合理水平的突变。在Omegon-KM内存在大肠杆菌特异的复制起点(ORI)，使得在大肠杆菌中快速和容易地克隆转座事件位点两侧的核苷酸序列。
To combine the features of the .OMEGA. interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the .OMEGA. interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (aphA) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transpostion. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.