• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Molecular and Cellular Biology >Efficient transformation of Dictyostelium discoideum amoebae.
【24h】

Efficient transformation of Dictyostelium discoideum amoebae.

机译：盘基网柄菌的高效转化。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.
机译：我们已经使用质粒pAG60的衍生物转化了盘基网柄菌，该质粒被设计用于转化哺乳动物细胞。该质粒带有与细菌基因kan相连的1型单纯疱疹病毒胸苷激酶基因的启动子区域，该基因编码氨基糖苷3'-磷酸转移酶。 kan来自Tn5转座子。磷酸转移酶的表达允许通过转化细胞对抗生素G-418的抗性直接选择。 pAG60不能转化D.discoideum，但是通过将D.discoideum序列克隆到四环素抗性基因中而使其转化熟练。大部分转化细胞正常生长和发育，并分化为G-418抗性孢子。这些转化子是不稳定的，并且在缺乏抗生素选择的情况下生长过程中会迅速失去对G-418的抗性。 Southern印迹表明，这些不稳定的G-418抗性转化体带有pAG322的pBR322和kan序列。 pAG60-D。用于转化的discoideum重组质粒的构建方法可能会使它们诱变。我们已经用pAG60-D的文库转化了D. discoideum后分离出了几个发育突变体。 Discoideum重组质粒。这些突变体具有G-418抗性，并在其核DNA中带有pAG60。我们回收了pAG60-D。 Discoideum重组质粒，来自几个发育突变体。该质粒以升高的频率转化迪斯科圆盘菌并整合到核基因组中。我们推测整合可以导致基因的插入失活，这对于分化而不是生长至关重要。仅当转化质粒与迪斯科圆盘菌核DNA具有同源性时，才会发生诱变转化。哺乳动物细胞转化载体pSV2-neo没有携带D. discoideum序列，并且能够转化。但是，pSV2-neo转化不是诱变的。这些结果表明，直接杀灭和恢复对于碟形梭状芽胞杆菌的分化必不可少的基因是可能的。

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