INTERPOSON MUTAGENESIS OF SOIL AND WATER BACTERIA - A FAMILY OF DNA FRAGMENTS DESIGNED FOR INVITRO INSERTIONAL MUTAGENESIS OF GRAM-NEGATIVE BACTERIA
INTERPOSON MUTAGENESIS OF SOIL AND WATER BACTERIA - A FAMILY OF DNA FRAGMENTS DESIGNED FOR INVITRO INSERTIONAL MUTAGENESIS OF GRAM-NEGATIVE BACTERIA
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DOI：
10.1016/0378-1119(87)90041-2
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发表时间：
1987-01-01
期刊：
影响因子：
3.5
通讯作者：
KRISCH, H
中科院分区：
文献类型：
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作者：
FELLAY, R;FREY, J;KRISCH, H
我们构造了一系列。omega的导数。间位子[Prentki and Krisch， Gene 29(1984) 303-313]可用于体外插入诱变。这些DNA片段中的每一个都携带不同的抗生素或Hg2+抗性基因（ApR, CmR, TcR， KmR或HgR），这些基因被转录和翻译终止信号以及合成多连接物以倒置的方向连接在两侧。这些间位子的DNA可以很容易地纯化，然后通过体外连接插入到由DNA酶I随机线性化或由限制性内切酶在特定位点线性化的质粒中。含有介子插入的质粒分子可以通过其耐药性的表达来鉴定。间子的位置可以通过侧翼多连接体中的限制性位点精确地映射出来。为了验证它们的属性，我们使用了这些。omega。衍生物诱变广泛的宿主质粒，该质粒包含恶臭假单胞菌降解甲苯质粒pWW0的整个元裂解途径。这些介子插入到启动子和儿茶酚2,3-双加氧酶（C230）基因之间的质粒中，显著降低了该酶在大肠杆菌中的表达。我们还表明，当一个质粒含有。omega。间位子通过偶联动员从大肠杆菌转移到恶臭杆菌、农杆菌、菊花Erwinia、反硝化副球菌或豆科根瘤菌，通常表达适当的间位子耐药性，并且与未突变的质粒相比，检测到的C230活性水平大大降低。因此，. omega的选择和/或特性。插入突变可以在这些细菌种类中进行。
We have constructed a series of derivatives of the .OMEGA. interposon [Prentki and Krisch, Gene 29 (1984) 303-313] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these .OMEGA. derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C230) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing in .OMEGA. interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C230 activity are detected. Thus, the selection and/or characterization of .OMEGA. insertional mutations can be carried out in these bacterial species.