In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors

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Title: In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors
Author: Nyberg, Tarah Michelle
Abstract: The splicing mechanism of group II introns is analogous to that of nuclear introns and it is generally thought that both share a common ancestor . This work contains two studies of group II intron splicing in yeast mitochondria . Previous studies done in collaboration with Dr . Anna Pyle at Yale identified several important determinants for in vitro branch -site selection of intron aI5gamma : the presence of a bulged A (A880 ) , the 5' flanking GU base pair and the branch location within domain VI . I confirmed the in vitro findings in vivo and show that displacing the branch adenosine by one nucleotide in either direction can support branching at the shifted bulged A in vivo . Returning the base -pairs flanking the shifted branch -points to GU pairs increased both the efficiency and fidelity of branching at the ectopic branch A . However , for the shifted down ectopic branch A , it is not the presence of the GU pair flanking the branch that restores branching but the presence of a GC pair located two base -pairs above the branch . This finding is consistent with our observations that for the wild -type branch location , the branch environment above and below the branch are distinct . It appears that the short stem below the branch is important for the second splicing step . The goal of the second project was to identify novel nuclear genes that are involved in mitochondrial intron splicing . Based on the yeast genome project and several recent proteomic studies of yeast mitochondria , we identified 808 nuclear genes coding for potential mitochondrial proteins that can be deleted without lethality . Of these , 476 deletion strains retain a complete copy of the mtDNA (13 introns ) and have a respiratory growth defect . Those strains were screened by northern blot analysis for intron splicing defects . I observed the expected splicing defects in strains deleted for MSS18 , CBP2 and PET54 . I observed a novel splicing pattern in strains deleted for IMP1 , CBS2 , PET111 , MNE1 , AAT1 , ATP10 and PIF1 .
URI: http : / /hdl .handle .net /2152 .5 /428
Date: 2006-08-11

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In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /428 .

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