• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment
【24h】

I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment

机译：通过随机诱变和顺序体外富集确定I-PpoI和I-CreI归巢位点序列的简并性

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Plasmid libraries containing partially randomized cleavage sites for the eukaryotic homing endonucleases I-PpoI and I-CreI were constructed, and sites that could be cleaved by I-PpoI or I-CreI were selectively recovered by successive cycles of cleavage and gel separation followed by religation and growth in Escherichia coli. Twenty-one different I-PpoI-sensitive homing sites, including the native homing site, were isolated. These sites were identical at four nucleotide positions within the 15 bp homing site, had a restricted pattern of base substitutions at the remaining 11 positions and displayed a preference for purines flanking the top strand of the homing site sequence. Twenty-one different I-CreI-sensitive homing sites, including the native site, were isolated. Ten nucleotide positions were identical in homing site variants that were I-CreI-sensitive and required the addition of SDS for efficient cleavage product release. Four of these ten positions were identical in homing sites that did not require SDS for product release. There was a preference for pyrimidines flanking the top strand of the homing site sequence. Three of the 24 I-CreI homing site nucleotide positions apparently lacked informational content, i.e. were permissive of cleavage when occupied by any nucleotide. These results suggest that I-PpoI and I-CreI make a large number of DNA-protein contacts across their homing site sequences, and that different subsets of these contacts may be sufficient to maintain a high degree of sequence-specific homing site recognition and cleavage. The sequential enrichment protocol we used should be useful for defining the sequence degeneracy and informational content of other homing endonuclease target sites. (C) 1998 Academic Press. [References: 36]
机译：构建了包含真核归巢内切核酸酶I-PpoI和I-CreI的部分随机切割位点的质粒文库，并通过连续的切割和凝胶分离然后再连接的循环，选择性地回收了可以被I-PpoI或I-CreI切割的位点。和大肠杆菌的生长。分离了21个不同的I-PpoI敏感归巢位点，包括本地归巢位点。这些位点在15 bp归巢位点内的四个核苷酸位置相同，在其余11个位置具有受限的碱基取代模式，并显示出对位于归巢位点序列顶部链两侧的嘌呤的偏爱。隔离了二十一个不同的I-CreI敏感归巢位点，包括本机位点。在对I-CreI敏感的归巢位点变体中，十个核苷酸位置相同，并且需要添加SDS才能有效裂解产物。这十个位置中的四个在不需要SDS产品发布的归位站点中是相同的。优选在归巢位点序列的顶部链侧翼的嘧啶。 24个I-CreI归巢位点核苷酸位置中的三个显然缺乏信息内容，即当被任何核苷酸占据时允许裂解。这些结果表明，I-PpoI和I-CreI在其归巢位点序列之间形成大量DNA-蛋白质接触，并且这些接触的不同子集可能足以维持高度的序列特异性归巢位点识别和裂解。我们使用的顺序富集协议应该用于定义其他归巢核酸内切酶靶位点的序列简并和信息含量。 （C）1998年学术出版社。 [参考：36]

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号