Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Methods Mol Biol. 2022;2421:103-114. doi: 10.1007/978-1-0716-1944-5_7.
Mutator enzymes alter the nucleotide sequences of DNA or RNA molecules; immune systems utilize them to destroy the integrity of pathogen genomes and to optimize immune mediators of the host. Their dysregulation has been linked to tumorigenesis in various tissues. Defining and comparing the activities of such mutator enzymes requires a robust versatile assay that is independent of their biological context as in vivo mutation rates are typically low. Here we provide detailed protocols for two widely used E. coli-based approaches that detect the activities of ectopically expressed cytidine deaminases on two distinct reporter genes: an extrachromosomal kanamycin-resistance gene or an endogenous chromosomal substrate, the rpoB gene-encoding RNA polymerase. The generation of mutations is in both cases measured in a colony formation assay. With appropriate modifications, these assays can be extended to study other mutator enzymes.
突变酶改变 DNA 或 RNA 分子的核苷酸序列；免疫系统利用它们破坏病原体基因组的完整性，并优化宿主的免疫介质。它们的失调与各种组织的肿瘤发生有关。定义和比较这种突变酶的活性需要一种稳健的多功能测定法，该方法不依赖于其生物背景，因为体内突变率通常较低。在这里，我们提供了两种广泛使用的基于大肠杆菌的方法的详细方案，用于检测异位表达的胞嘧啶脱氨酶在两个不同报告基因上的活性：一个染色体外的卡那霉素抗性基因或一个内源性染色体底物，编码 RNA 聚合酶的 rpoB 基因。在两种情况下，突变的产生都在集落形成测定中进行测量。通过适当的修改，这些测定可以扩展到研究其他突变酶。
