Nuclear genes that promote splicing of the chloroplast group-I 23S rRNA intron and an organelle intron database, FUGOID
Cr. LSU is a self-splicing Group I intron in the chloroplast 23S rRNA gene of the green alga Chlamydomonas reinhardtii. Trans-acting factors are likely required to promote the splicing of this intron in vivo; however, nothing is known of the trans requirements for Group I intron splicing in chloroplasts. I have used a genetic approach in this study of possible splicing factors for Cr. LSU. Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the intron. In vitro assays showed that these substitutions had very strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be partially recovered by vi increasing the Mg2+ concentration, indicative of structural effects. The mutant constructs were transformed into chloroplasts to replace the wild-type intron. Surprisingly, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4 125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least 2 nuclear genes. The 7151 suppressor, which defines the css1 (chloroplast splicing suppressor) gene, was shown to have no discernible phenotype with the wild-type intron, and to be dominant in vegetative diploids containing the mutant intron. These results indicate that the Cr.LSU intron is unusually sensitive to single base changes in the core, and, moreover, provide the first identification of plant genes that promote splicing of a group I intron in vivo. Toward the eventual cloning of the css1 gene, a library was constructed in a novel cosmid vector, SCBN, using genomic DNA from the 7151 suppressor mutant of C. reinhardtii. Part of the library was used to generate an indexed collection of 10,340 individual clones in 110 96-well microtiter plates. The library can be easily replicated (for DNA preparation) and has an average insert size of ~37 kb. Thus, it covers almost 4 genome equivalents. The library may allow the cloning of css1 by complementation of the P4 125A mutant. Finally, I have developed a web-based relational database, FUGOID (Functional Genomics of Organellar Introns Database) that collects and integrates vii viii various functional (and some structural) data on organellar (mitochondrial and chloroplast)introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. Users can access the database with any common web browser on a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm.