Unexpected instability of family of repeats (FR), the critical cis-acting sequence required for EBV latent infection, in EBV-BAC systems.

Unexpected instability of family of repeats (FR), the critical cis-acting sequence required for EBV latent infection, in EBV-BAC systems.
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DOI:
10.1371/journal.pone.0027758
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发表时间:
2011
期刊:
影响因子:
3.7
通讯作者:
Tsurumi T
Tsurumi T
中科院分区:
综合性期刊3区
文献类型:
--
作者:
Kanda T;Shibata S;Saito S;Murata T;Isomura H;Yoshiyama H;Takada K;Tsurumi T

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一组被称为重复序列家族(FR)的重复序列是EBV潜伏感染所需的关键顺式作用序列。FR序列在EB病毒株间是异质的,当在E.基于大肠杆菌的克隆载体。然而,在EBV-BAC(细菌人工染色体)系统中FR的稳定性尚未被研究。我们发现Akata株EBV的全长FR不能稳定地保持在BAC载体中。相比之下,新获得的EBV B 95 -8株的BAC克隆在重组病毒生产和B细胞转化期间稳定地保持全长FR。对Akata来源的EBV-BAC克隆的一级DNA序列的研究表明,FR不稳定性最有可能是由于FR区的推定二级结构。我们的结论是,在EBV-BAC克隆FR的不稳定性可能是一个陷阱,在E。大肠杆菌介导的EBV遗传学。
A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akata–derived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics.
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