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 i s 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.
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