Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2021;2167:3-11. doi: 10.1007/978-1-0716-0716-9_1.
Group II introns are noncoding sequences that interrupt genes, and that must be removed or spliced-out at the RNA level during gene expression. Following the transcription of interrupted genes, group II introns self-splice while concurrently ligating their flanking exons to generate mature mRNAs ready for translation. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, interrupts the gene coding for a relaxase enzyme that initiates the transfer of mobile elements by conjugation. This functional link between group II intron splicing and conjugative transfer enabled us to engineer highly sensitive splicing assays using the native biological context of Ll.LtrB. The splicing efficiency/conjugation assay was developed to determine the splicing competence of various Ll.LtrB mutants, whereas the splicing selection/conjugation assay was established to isolate splicing-proficient variants from a randomly generated bank of mutated introns.
内含子 II 是一种非编码序列，会打断基因，并且在基因表达过程中必须在 RNA 水平上被移除或剪接。在被打断的基因转录之后，内含子 II 会自我剪接，同时将其侧翼的外显子连接起来，生成准备翻译的成熟 mRNA。来自革兰氏阳性菌乳球菌乳亚种的模型内含子 II Ll.LtrB 打断了编码松弛酶的基因，而松弛酶是通过接合作用启动移动元件转移的酶。这种内含子 II 剪接和接合转移之间的功能联系使我们能够利用 Ll.LtrB 的天然生物学背景来设计高度敏感的剪接测定。剪接效率/接合测定用于确定各种 Ll.LtrB 突变体的剪接能力，而剪接选择/接合测定则用于从随机产生的突变内含子库中分离出剪接有效的变体。
