Cann Isaac K O, Amaya Kensey R, Southworth Maurice W, Perler Francine B
New England Biolabs, Inc, Beverly, Massachusetts 01915, USA.
Appl Environ Microbiol. 2004 May;70(5):3158-62. doi: 10.1128/AEM.70.5.3158-3162.2004.
A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.
基于用条件性无活性DNA聚合酶拯救T4噬菌体菌株的能力，开发了一种检测内含肽剪接和非剪接活性的遗传选择系统。这种噬菌体缺陷可以通过质粒编码的噬菌体RB69 DNA聚合酶的表达来互补。将内含肽基因插入RB69 DNA聚合酶基因的活性位点，使聚合酶活性和噬菌体活力依赖于蛋白质剪接。通过筛选热敏剪接突变体来测试该系统的有效性。开发具有识别蛋白质剪接抑制剂潜力的遗传系统是控制在必需蛋白质中携带内含肽的致病微生物增殖的第一步。
