SITE-SPECIFIC RECOMBINATION BY GIN OF BACTERIOPHAGE-MU - INVERSIONS AND DELETIONS
SITE-SPECIFIC RECOMBINATION BY GIN OF BACTERIOPHAGE-MU - INVERSIONS AND DELETIONS
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DOI：
10.1016/0042-6822(83)90367-7
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发表时间：
1983-01-01
期刊：
影响因子：
3.7
通讯作者：
VANDEPUTTE, P
中科院分区：
文献类型：
--
作者：
PLASTERK, RHA;ILMER, TAM;VANDEPUTTE, P
噬菌体Mu DNA中的3000 [碱基对]可逆片段决定了噬菌体的宿主范围。反转由噬菌体编码的蛋白质Gin催化;重组位点是短的反向重复序列。杜松子酒蛋白质只由Mu少量生产。为了进一步研究Gin介导的重组反应，构建了Gin高产菌株。将gin基因克隆在噬菌体pL启动子后面的质粒上。这导致Mu gin噬菌体的倒位频率比Gin从其自身启动子表达的情况高100倍。开发了一种适用于体内和体外检测Gin作用的测试系统：将大肠杆菌的lacZ基因克隆到可逆区域内，使得其仅在该区域处于一个特定方向时表达。因此，可以检测或选择反转作为从Lac-到Lac+的切换。利用该系统测定了不同实验条件下的反转频率。通过使用限制性内切酶在体外反转2个重组位点中的1个，并对这些位点之间的DNA进行遗传标记，研究了Gin催化缺失的能力。删除确实会发生，尽管频率低于倒位。
A 3000 [base-pair] bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the pL-promoter of phage .lambda.. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of Escherichia coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus, inversions can be detected or selected as a switch from Lac- to Lac+. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro 1 of the 2 recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.