GENETIC AND MOLECULAR STUDIES OF GROUP II INTRONS

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Splicing and genetic properties of group II introns will be investigated in mitochondria of bakers yeast, Saccharomyces cerevisiae. Introns I and 2 of the COXI gene of mtDNA code for a protein with reverse transcriptase (RT), endonuclease and maturase (splicing) functions essential for the splicing and homing/transposition by the introns. Both introns are highly efficient, site-specific retroelements clearly related to non-LTR- retrotransposons, including the LINE elements of the human genome. Major and minor intron homing pathways have been identified, all of which depend on a remarkable reaction in which a complex containing the intron-encoded RT and excised intron RNA lariat inserts the intron RNA into the sense strand of the intron-less DNA target site by a reverse splicing reaction. Subsequent steps vary widely among various RT- independent pathways. Bakers yeast has numerous attributes that make it a powerful and facile system for research on mechanisms of group II intron homing, and for the mechanisms of site-specific homing by group II introns to obtain a clear view of how the various pathways differ and, or importantly, to show how a common homing intermediate is partitioned among processes, transposition was likely a mechanism by which introns spread during evolution. A system will be developed to detect retrotransposition of a tagged intron from mitochondria to the nucleus. The third aim is to characterize aspects of group II intron molecular biology. The main emphasis is to identify nuclear will analyze genes that process the precursor to the intron-encoded RT, control the stability and degradation of excised group II intron RNAs, and assist the self-splicing of the introns. Finally, the RNA binding and splicing functions of the naturase domain of the intron-encoded protein will be studied.