CHLOROPLAST PRE-MRNA SPLICING: GENETICS AND BIOCHEMISTRY

这一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。 藻类基因有四个大内含子，其中至少两个内含子包含 大小显著但未知的开放阅读框(ORF) 功能。莱茵哈迪提非常适合于遗传分析。 光合作用基因：亲本叶绿体的重组 异体交配过程中的基因组允许遗传 叶绿体基因图谱，以及这种有机体 以醋酸盐为唯一碳源生长，可供选择 以及光合作用突变体的维持。叶绿体突变体 将识别和表征PSBA拼接中的缺陷 从基因和生化角度来说。我们的目标是通过 可能的顺显性和反隐性PSBA的鉴定 突变，主要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参与 剪接过程中的蛋白质。身份识别和 任何影响PSBA剪接的核突变体的特征 将有助于建立核因子在加工过程中的作用 叶绿体RNA。野生型细胞以及核和 叶绿体突变体的特征如下：1) RNA在Northern blotts上的剪接模式及其稳定性 剪接内含子，2)内含子在体外自剪接的能力， 和与这种活性相关的核苷酸序列，3)细胞 与内含子ORF对应的蛋白质的位置和存在， 4)蛋白质在大肠杆菌中可能的酶活性 编码于PSBA内含子ORF。

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.