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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Rescue and characterization of episomally replicating DNA from the moss Physcomitrella
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Rescue and characterization of episomally replicating DNA from the moss Physcomitrella

机译：苔藓假单胞菌的复制复制DNA的营救和鉴定

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摘要

The moss Physcomitrella is unique among plants in that it permits efficient gene targeting by homologous recombination. Furthermore, transformed DNA can replicate episomally in Physcomitrella. Here we show that episomally replicating DNA can be rescued back into Escherichia coli, and we use such rescue to study the fate of the transformed DNA. Significantly, plasmids rescued from moss transformed with circular DNA are identical to the original plasmid, whereas plasmids rescued from moss transformed with linearized DNA frequently have deletions created by direct repeat recombination. These events are highly predictable in that they target the longest direct repeat on the plasmid if this repeat is at least 12 bp. Episomal transformants obtained with linearized DNA show a more than 1,000-fold amplification of the DNA whereas transformants obtained with circular DNA have much lower copy numbers. Most episomal transformants quickly lose the plasmid in the absence of selection, but a semistable type of transformant that loses the plasmid at a much lower frequency was also observed. The consistent rescue of the original plasmid, or of predictable derivatives thereof, suggests that molecular genetics methods which rely on shuttle plasmids are feasible in Physcomitrella.
机译：苔藓假单胞菌在植物中是独特的，因为它允许通过同源重组进行有效的基因靶向。此外，转化的DNA可以在假单胞菌中游离复制。在这里，我们证明了可以将游离复制的DNA挽回回到大肠杆菌中，并且我们使用这种挽救来研究转化的DNA的命运。重要的是，从用环状DNA转化的苔藓中拯救的质粒与原始质粒相同，而从用线性化DNA转化的苔藓中拯救的质粒通常具有通过直接重复重组产生的缺失。这些事件是高度可预测的，因为如果该重复序列至少为12 bp，它们将靶向质粒上最长的直接重复序列。用线性化DNA获得的游离型转化体显示出超过1000倍的DNA扩增，而用环状DNA获得的转化体具有低得多的拷贝数。在没有选择的情况下，大多数游离型转化子会很快丢失质粒，但也观察到以低得多的频率丢失质粒的半稳定型转化子。对原始质粒或其可预测衍生物的一致挽救表明，依赖穿梭质粒的分子遗传学方法在Physcomitrella中是可行的。

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  • 作者

    Eva Muren; Anders Nilsson; Mikael Ulfstedt; Monika Johansson; Hans Ronne;

  • 作者单位

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden;

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden;

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden;

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden Department of Food Science, Swedish University of Agricultural Sciences, Box 7051, SE-750 07 Uppsala, Sweden;

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    episomal replication; plasmid rescue; recombination; shuttle plasmid;

    机译：游离复制;质粒拯救;重组穿梭质粒;

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