Pneumocystis carinii is the major infectious cause of fatal complications in AIDS patients. All characterized isolates of this opportunistic pathogen harbor one or two group I self-splicing intron ribozymes in their chromosomal rRNA genes, which must excise themselves from the primary transcripts of these genes in order to generate mature rRNA. Since such introns are not found in nuclear genomes of humans, agents inhibiting group I intron splicing are potential anti-Pneumocystis agents. We have found that a variety of agents, including derivatives of arginine and pentamidine inhibit the catalytic activity of these group I introns in vitro. Since P. carinii cannot be cultured or genetically characterized, we plan to generate surrogate organisms for studying the effect of intron splicing inhibition in vivo by inserting the P. carinii introns into 1acZ and the rRNA genes of the yeast Saccharomyces cerevisiae, which is phylogenetically related to P. carinii. The yeast strains harboring these introns will be used as surrogate strains to test the ability of splicing inhibitors to block splicing in intact cells and to inhibit cell growth if those cells contain introns in essential genes. The intron containing yeast strains will be used to generate intron an nuclear gene mutants which are resistant to inhibitors. Thee mutants will be characterized to determine the intron sites required for inhibitor sensitivity and the cellular genes which may be required for entry of the inhibitors. The intron mutants will also be compared with mutants generated by ex vivo evolution of isolated introns, and with the products of site-directed mutagenesis, in order to define the target on which the inhibitors act. The surrogate strains will also be used to isolate splicing-defective mutants. Splicing defective mutations mapping to the intron will help to define the intron residues which are essential for splicing. Mutations mapping at other nuclear genes will define transacting factors which may facilitate splicing and which may represent future targets for the development of chemotherapeutic agents. This project apply the methods of molecular genetics to the splicing of nuclear group I introns, which have not been extensively studied in this way since they do not naturally occur in organisms as genetically tractable as yeast.
