公开(公告)号：US5491060A

公开(公告)日：1996-02-13

申请号：US956578

申请日：1992-10-06

申请人： Daniel C. Stein ,  Andrejez S. Piekarowicz ,  Robert T. Yuan

发明人： Daniel C. Stein ,  Andrejez S. Piekarowicz ,  Robert T. Yuan

IPC分类号： C12N9/10 ,  C12N9/22 ,  C12N15/54 ,  C12N15/70 ,  C12Q1/68 ,  C12N15/09 ,  C12N15/55

CPC分类号： C12N9/22 ,  C12N15/70 ,  C12N9/1007

摘要： E. coli bacterial strains encoding a restriction gene that degrades methylated DNA, and prevents cloning of genes expressing the methyltransferase responsible for methylation, are mutated by a chemical or physical mutagen, so as to make the restriction enzyme temperature sensitive. Mutant cells are rendered competent, and plasmids expected or known to contain genes encoding methyltransferase enzymes are introduced. The transformants grow at the permissive temperature, where the restriction enzyme system is inactivated due to the mutated gene. Successful clones, expressing a methyltransferase, can be quickly identified by those which grow at the permissive temperature, but not at the non-permissive temperature. The valuable methyltransferases, as well as restriction enzymes associated therewith, can accordingly be recovered in large quantity.

摘要翻译： 通过化学或物理诱变剂突变编码降解甲基化DNA的限制性基因并防止克隆表达甲基化的甲基转移酶的基因的大肠杆菌细菌菌株，使得限制酶温度敏感。 引入突变体细胞，并引入预期或已知含有编码甲基转移酶基因的质粒。 转化体在允许的温度下生长，其中限制酶系统由于突变的基因而失活。 表达甲基转移酶的成功克隆可以通过在许可温度下生长而不是在非允许温度下生长的那些快速鉴定。 因此，可以大量回收有价值的甲基转移酶以及与其相关的限制酶。