蓝细菌断裂DNA聚合酶DnaE的体内/外反式剪接分析

蓝细菌Anabaena PCC7120的DNA聚合酶DnaE由2个断裂基因dnaENI和dnaECI编码.通过纯化它们的部分基因在大肠杆菌中的表达产物DnaENI′和DnaECI′来免疫家兔获得抗体,用于对断裂的DnaE在体内和体外的反式剪接活性进行鉴定.在体外实验中,将2个断裂的DNA聚合酶DnaENI′和DnaECI′混合,进行反应,并利用它们的抗体对反应产物进行鉴定;在体内实验中,利用以上抗体对Anabaena PCC7120细胞总蛋白进行免疫杂交分析.实验结果表明,蓝细菌AnabaenaPCC7120中断裂的2个DnaE多肽在体内和体外均能进行反式剪接.体外实验中,纯化的DnaENI′和DnaECI′的混合反应产生新的蛋白产物,既有成熟的反式剪接产物DnaEN′-,又有剪切了内含肽后的副产物DnaEN′和DnaE,且DTT的存在对剪接反应具有促进作用;此外体内实验中,在Anabaena PCC7120细胞中既能检测到完整的DnaE,也能检测到未作用的DnaENI,但未能检测到DnaECI.

Analysis of Trans-splicing of Split DNA Polymerase DnaE in Cyanobacterium

We splitted the DNA polymerase DnaE of cyanobacterium Anabaena PCC 7120 into two coding fragments, dnaENI and dnaECI,and expressed in Escherichia coli. The antisera against the encoded products, DnaENI′ and DnaECI′ were raised by injecting rabbits with purified proteins. The products of the trans-splicing reaction were determined with the DnaENI′ and DnaECI′ antibodies. The immunostaining of the DnaENI and DnaECI expression in Anabaena PCC 7120 was also carried out. By mixing purified DnaENI′ and DnaECI′, mature trans-splicing product DnaEN′-C′, as well as the intein-cleaved products DnaEN′ and DnaEC′. We also found that dithiothreitol (DTT) promoted the efficiency of trans-splicing. Both intact DnaE and native DnaENI proteins could be detected in Anabaena PCC 7120, but not DnaECI.