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The 4.1 Mb of targeted DNA included the 16 kb mtDNA and all coding and untranslated exons of 1381 nuclear genes, including 1013 mitochondrial genes from the MitoCarta database (Pagliarini et?al., 2008), 21 genes with recent strong evidence of mitochondrial association, and 347 additional genes. All analyses were restricted to the mtDNA and coding exons of the 1034 genes with confident evidence of mitochondrial association (1.4?Mb). Detailed methods for target selection, sequencing, alignment and variant detection are submitted elsewhere (S.E.C., unpublished data). Differences in DNA sequence between each individual and the GRCh37 human reference assembly were identified. check details Nuclear variants that passed quality control metrics were prioritized according to three criteria: (1) SNV allele frequency Selleck Anti-infection Compound Library and was cloned into the 4-hydroxytamoxifen-inducible lentiviral vector, pF_5x_UAS_MCS_SV40_puroGEV16-W ( Yeap et?al., 2010). MTFMT viral particles were generated and patient fibroblasts were transduced as described previously ( Calvo et?al., 2010). Three independent transductions were performed and cells were harvested after 10�C12?days selection with 1?��g/ml puromycin. Total RNA was isolated from frozen cells under acidic conditions with TRIzol (Invitrogen) according to the manufacturer's instructions. Acid-washed glass beads (0.5?mm diameter; Sigma) were added during extraction. Total RNAs were separated by acid-urea PAGE as described previously (Enr��quez and Attardi, 1996, K?hrer and RajBhandary, ATP7A 2008?and?Varshney et?al., 1991) with modifications. In brief, 0.1 A260 units of each RNA sample were loaded onto a 6.5% polyacrylamide gel containing 7?M urea and 0.2?M sodium acetate (pH 5.0). Individual tRNAs were detected by northern blotting (K?hrer and RajBhandary, 2008) with the following hybridization probes: 5��TAGTACGGGAAGGGTATAA3�� (mitochondrial tRNAMet) and 5��TTCCACTGCACCACTCTGCT3�� (cytoplasmic initiator tRNAiMet). Northern blots were quantified by PhosphorImager analysis with ImageQuant software (Molecular Dynamics). Experiments were performed in duplicate.