SITE-SPECIFIC DELETIONS OF CHROMOSOMALLY LOCATED DNA SEGMENTS WITH THE MULTIMER RESOLUTION SYSTEM OF BROAD-HOST-RANGE PLASMID RP4
SITE-SPECIFIC DELETIONS OF CHROMOSOMALLY LOCATED DNA SEGMENTS WITH THE MULTIMER RESOLUTION SYSTEM OF BROAD-HOST-RANGE PLASMID RP4
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DOI：
10.1128/jb.177.1.52-58.1995
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发表时间：
1995-01-01
影响因子：
3.2
通讯作者：
DELORENZO, V
中科院分区：
文献类型：
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作者：
KRISTENSEN, CS;EBERL, L;DELORENZO, V
广泛宿主范围质粒RP 4的多聚体解析系统（mrs）已被开发用于开发一种通用方法，该方法允许在多种革兰氏阴性细菌中精确切除染色体片段。该方法基于RP 4的两个直接重复的140-bp分辨（res）序列之间的位点特异性重组，该重组受由帕拉基因编码的质粒携带的分辨酶的影响。用杂交的mini-Tn 5转座子监测mrs系统删除染色体两侧有res位点的部分的效率和准确性，在该转座子中，各种颜色的转座子被插入到质粒载体中。（β-半乳糖苷酶和儿茶酚2，3双加氧酶）或发光将与res序列相关的（哈氏弧菌荧光素酶）表型标记插入靶细菌的染色体中，并在体内暴露于帕拉基因的产物。获得了高频率的标记切除，帕拉表达系统的不同构型表明仅需要少数解离酶分子来实现位点特异性重组事件，从不能在靶细菌中复制的质粒瞬时表达帕拉有助于在插入假单胞菌染色体中的复杂杂合转座子内实现差异缺失putida，这种策略允许异源DNA片段的稳定遗传，这些片段实际上缺乏最初用于选择其插入的序列。
The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene, The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene, The high frequencies of marker excision obtained, with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event, Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida, This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.