Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo
Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo
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DOI：
10.1073/pnas.93.22.12400
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发表时间：
1996-10-29
影响因子：
11.1
通讯作者：
Johansen, IE
中科院分区：
文献类型：
--
作者：
Johansen, IE
将内含子插入到两个植物马铃薯Y病毒豌豆种子传花叶病毒分离物的cDNA中，促进了在大肠杆菌中的质粒扩增。插入的内含子中的多个终止密码子干扰了病毒基因的开放阅读框，从而终止了病毒蛋白在大肠杆菌中的不良翻译，含有全长病毒序列的质粒在花椰菜花叶病毒35S启动子和诺帕林合成酶终止信号的控制下，在大肠杆菌中稳定且易于扩增。如果将一个或多个内含子插入到病毒序列中，这些质粒在机械接种到豌豆叶片上时是有侵染性的，对cDNA源病毒的检测证实，体内转录的前-mRNA的内含子剪接如预期的那样发生了，重建了病毒基因组序列。克隆病毒和亲本病毒的症状发展和病毒积累是一致的。有人认为，内含子插入可用于操作和扩增在大肠杆菌中不稳定或有毒的克隆DNA片段。当在真核细胞中体内转录时，内含子将从序列中消除，不会干扰进一步的蛋白质表达分析或病毒感染。
Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli, Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence, These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves, Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.