Breul A, Kuchinke W, von Wilcken-Bergmann B, Müller-Hill B
Institut für Genetik, Universität zu Köln, Federal Republic of Germany.
Eur J Biochem. 1991 Jan 1;195(1):191-4. doi: 10.1111/j.1432-1033.1991.tb15694.x.
Synthetic octameric oligonucleotides that code for a unique restriction site were cloned into a randomly linearized plasmid that carries the lacZ gene. The insertions were mapped by digestion with appropriate restriction endonucleases. 12 mutants were identified which carry an insertion within the lacZ gene and still express active beta-galactosidase. Small deletions or duplications of the wild-type sequence occurred at these positions which restore the correct reading frame. The insertions occurred in the first and the last third of the internal duplication of the lacZ gene and within the domain homologous to dihydrofolate reductase.
编码一个独特限制位点的合成八聚体寡核苷酸被克隆到携带lacZ基因的随机线性化质粒中。通过用适当的限制性内切酶消化来对插入片段进行定位。鉴定出12个突变体，它们在lacZ基因内有一个插入并且仍表达活性β-半乳糖苷酶。在这些位置发生了野生型序列的小缺失或重复，从而恢复了正确的阅读框。插入发生在lacZ基因内部重复的前三分之一和后三分之一以及与二氢叶酸还原酶同源的结构域内。
