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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Characterization of the lactococcal group II intron target site in its native host
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Characterization of the lactococcal group II intron target site in its native host

机译：乳球菌II型内含子靶位点在其天然宿主中的表征

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摘要

The Lactococcus lactis group II intron (LI.ItrB) retrohomes into the ltrB gene at high efficiency. To date, the critical DNA bases recognized in vivo by the Ll.ltrB ribonucleoprotein (RNP) have been exclusively elucidated in Escherichia coli. However, recent evidence indicates host-dependant differences in Ll.ltrB mobility, raising the possibility of limitations of the current model for RNP-homing site recognition in the native L. lactis host. In this work, intron retargeting experiments in L. lactis have demonstrated that adherence to specific target site critical bases is not sufficient to predict success or failure of chromosomal invasion, as in E. coli. Accordingly, a quantitative real-time PCR (QPCR) assay was developed to test target site nucleotides previously demonstrated as critical for homing in E. coli, for relevance in its native host. This two-plasmid QPCR homing assay is highly sensitive and, unlike previous E coli-based assays, resolves differential homing efficiencies in the absence of selection. As in E coli, deviation from wild type at target site positions -23, -21, -20, -19, and +5 resulted in lower homing efficiencies in L. lactis. Furthermore, the same trends are observed when assaying select variants in Enterococcus faecalis. Our results suggest that these target site positions are critical in both E coli and L. lactis. (c) 2007 Elsevier Inc. All rights reserved.
机译：乳酸乳球菌II组内含子（LI.ItrB）高效归巢到ltrB基因中。迄今为止，在大肠杆菌中已经专门阐明了在L.ltrB核糖核蛋白（RNP）体内识别的关键DNA碱基。但是，最近的证据表明，Ll.ltrB迁移率依赖于宿主，这增加了在天然乳酸乳球菌宿主中限制RNP归巢位点的当前模型的可能性。在这项工作中，在乳酸乳球菌中进行的内含子重靶向实验已经证明，对特定靶位点关键碱基的粘附不足以预测染色体入侵的成功或失败，就像在大肠杆菌中一样。因此，开发了定量实时PCR（QPCR）测定法以测试先前证明对于在大肠杆菌中归巢至关重要的靶位点核苷酸，以检测其天然宿主的相关性。这种两质粒QPCR归巢测定法非常灵敏，与以前的基于大肠杆菌的测定法不同，它可以在没有选择的情况下解决差异化的归巢效率。如在大肠杆菌中，在靶位点位置-23，-21，-20，-19和+5处偏离野生型导致乳酸乳球菌的归巢效率降低。此外，当在粪肠球菌中检测选择的变体时，观察到相同的趋势。我们的结果表明，这些靶位点位置在大肠杆菌和乳酸乳球菌中均至关重要。 （c）2007 Elsevier Inc.保留所有权利。

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