Waring R B, Ray J A, Edwards S W, Scazzocchio C, Davies R W
Cell. 1985 Feb;40(2):371-80. doi: 10.1016/0092-8674(85)90151-5.
We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.
我们利用大肠杆菌作为宿主，开发了一种用于嗜热四膜虫自我剪接核糖体RNA内含子的体内RNA剪接检测方法。一个包含内含子序列的DNA片段已被克隆到M13mp83中，使得β-半乳糖苷酶α片段的表达依赖于从mRNA前体中切除内含子。噬菌斑表型与切除的内含子RNA水平密切相关。通过寡核苷酸定向诱变在保守序列P、Q和S中引入点突变。所有突变都显示剪接减少，这与S的线粒体遗传数据一致，并首次直接证明P和Q在功能上很重要。结果支持了R与S以及P与Q的碱基配对对内含子结构和功能很重要的假说。
