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.
