Giza P E, Schmit D M, Murr B L
Gene. 1981 Dec;15(4):331-42. doi: 10.1016/0378-1119(81)90176-1.
Techniques were developed to mutagenize a single DNA strand in a specific region of the tetracycline-resistance (tetr) gene of the plasmid pKB280 that also carries the lambda repressor gene. Separate annealings of complementary single strands gave two isomeric, circular plasmids containing a 275-nucleotide, single-stranded region (gap) in the tetr gene. One of the isomeric, gapped plasmids was mutagenized specifically with sodium bisulfite such that an estimated 98% of the molecules had suffered at least one C to U conversion in the gap. The mutagenized gap was filled in with DNA polymerase. These molecules transformed Escherichia coli strain MM294 to lambda-immunity with the same frequency as unmutagenized, gap-filled pKB280. Of the lambda-immune transformants, 32% were Tcr and 68% were Tcs. Restriction analysis of plasmids from some Tcs transformants showed losses of restriction sites within the gap and at the gap termini, but none outside the gap. No deletions were detected.
已开发出一些技术，用于在质粒pKB280的四环素抗性（tetr）基因的特定区域诱变单条DNA链，该质粒还携带λ阻遏基因。互补单链的单独退火产生了两个异构的环状质粒，它们在tetr基因中含有一个275个核苷酸的单链区域（缺口）。其中一个异构的带缺口质粒用亚硫酸氢钠进行了特异性诱变，使得估计98%的分子在缺口中至少发生了一次C到U的转换。用DNA聚合酶填补诱变后的缺口。这些分子将大肠杆菌MM294菌株转化为λ免疫的频率与未诱变、填补缺口的pKB280相同。在λ免疫转化体中，32%是Tcr，68%是Tcs。对一些Tcs转化体的质粒进行限制性分析表明，缺口内和缺口末端的限制性位点缺失，但缺口外没有。未检测到缺失。
