CHLOROPLAST PRE-MRNA SPLICING: GENETICS AND BIOCHEMISTRY

这项建议结合了遗传学和分子生物学的工具， 生物学探索叶绿体RNA剪接的机制， 利用莱茵衣藻psbA基因编码， 光系统II的32kD除草剂结合蛋白。 的 藻类基因有四个大的内含子，其中至少有两个含有 重要但未知的开放阅读框（ORF） 功能 C. 莱茵衣藻非常适合于 光合基因：亲本叶绿体重组 在异宗配合交配过程中， 叶绿体基因图谱，以及这种生物体的能力， 以乙酸盐作为唯一碳源生长， 和光合突变体的维持。 叶绿体突变体 psbA剪接缺陷将被鉴定和表征 从基因和生物化学上。 我们的目标是通过 鉴定可能顺式显性和反式隐性psbA 突变，一级核苷酸序列的重要性 内含子剪接，以及可能的内含子编码的 蛋白质在剪接过程中 识别和 影响psbA剪接的任何核突变体的表征 将有助于确立核因子在处理 叶绿体RNA。 野生型细胞以及核和 叶绿体突变体的特征在于：1） RNA在北方印迹上的剪接模式和 剪接的内含子，2）内含子在体外自我剪接的能力， 和与该活性相关的核苷酸序列，3）细胞的 对应于内含子ORF的蛋白质的位置和出现， （4）E.蛋白质大肠杆菌 在psbA内含子ORF中编码。

This proposal combines the tools of genetics and molecular biology to explore the mechanism of chloroplast RNA splicing, making use of the Chlamydomanas reinhardtii psbA gene coding for the 32 kD herbicidebinding protein of Photosystem II. The algal gene has four large introns, at least two of which contain open reading frames (ORFs) of significant size but unknown function. C. reinhardtii is well-suited for a genetic analysis of photosynthetic genes: recombination of parental chloroplast genomes during heterothallic mating allows for the genetic mapping of chloroplast genes, and the ability of this organism to grow on acetate as a sole carbon source allows for the selection and maintenance of photosynthetic mutants. Chloroplast mutants defective in psbA splicing will be identified and characterized genetically and biochemically. The goal is to understand, through identification of possible cis-dominant and trans-recessive psbA mutations, the importance of the primary nucleotide sequence on intron splicing, and the involvement of possible intron-encoded proteins in the process of splicing. Identification and characterization of any nuclear mutants that affect psbA splicing will help establish a role for nuclear factors in processing chloroplast RNA. Wildtype cells as well as nuclear and chloroplast mutants will be characterized with respect to 1) the pattern of RNA splicing on Northern blots and the stability of spliced introns, 2) the ability of the introns to selfsplice in vitro, and nucleotide sequences pertinent to this activity, 3) the cellular location and occurrence of proteins corresponding to intron ORFs, and 4) the possible enzymatic activity in E. coli of proteins encoded in the psbA intron ORFs.