GENETIC AND MOLECULAR STUDIES OF RNA SPLICING

线粒体II类内含子oxi 3 I5 g的自我剪接机制， 将使用遗传学， 生物化学和重组DNA方法。 线粒体剪接缺陷 将分离该内含子的突变体。 它们的体内表型和 序列改变将被确定，并且它们在体外条件下的活性 确定了自剪接条件。 它们的假回复突变体将是 分离并进行类似分析。 内含子的进一步改变 将使用体外诱变方法进行序列测定， 并扩展对从研究中获得的功能重要序列的见解， 的体内突变体，并提供一个更完整的看法， 序列的 一种lacZ融合基因，其中β-半乳糖苷酶的表达 活性E.大肠杆菌细胞将依赖于内含子剪接， 作为一种提高效率的手段， 筛选内含子的体外突变。 核基因参与了 oxi 3/15 g的体内剪接将使用以下核抑制剂来鉴定： 线粒体剪接缺陷突变体和PET突变体。 主要重点 将给予的发展，在体外测定线粒体 参与I类和II类内含子剪接的蛋白质; 将研究每个类别的代表性内含子。 核蛋白 将对来自线粒体的颗粒（RNP）进行表征和测试， 它们促进内源性前体mRNA剪接的能力。 无rna 蛋白质样品，直接从线粒体分离或获得 在核酸酶处理分离的RNP后，将进行分级和测试 因为它们能够促进外源模型前体mRNA的剪接。 线粒体和核突变体的提取物将用于 识别参与剪接的特定蛋白质。

The mechanism of the self-splicing class II mitochondrial intron, oxi3 I5g, of the yeast, Saccharomyces cerevisiae will be studied using genetic, biochemical and recombinant DNA methods. Mitochondrial splicing defective mutants of that intron will be isolated. Their in vivo phenotypes and sequence alterations will be determined and their activity under in vitro self-splicing conditions determined. Pseudorevertants of them will be isolated and analyzed similarly. Further alterations of the intron sequence will be made using in vitro mutagenesis methods both to confirm and extend insights to functionally important sequences gained from studies of the in vivo mutants and to provide a more complete view of those sequences. A lacZ fusion gene, in which the expression of B-galactosidase activity in E. coli cells will depend on intron splicing, will be constructed and investigated as a means of enhancing the efficiency of screening in vitro mutations of the intron. Nuclear genes involved in the in vivo splicing of oxi3 I5g will be identified using nuclear suppresors of mitochondrial splicing defective mutants and pet- mutants. Major emphasis will be given to the development of an in vitro assay for mitochondrial proteins which participate in the splicing of class I and class II introns; representative introns of each class will be studied. Ribonucleoprotein particles (RNPs) from mitochondria will be characterized and tested for their ability to promote the splicing of endogenous pre-mRNAs. RNA-free protein samples, either isolated directly from mitochondria or obtained upon nuclease treatment of isolated RNPs, will be fractionated and tested for their ability to promote the splicing of exogenous model pre-mRNAs. Extracts from both mitochondrial and nuclear mutants will be used to identify specific proteins involved in splicing.