Figure 4.

Screening oTFAM chimeras for their ability to support replication of hmtDNA. (A,B), PCR genotyping strategies for cells expressing oTFAM chimeras. (C,D), PCR genotyping strategies for cells expressing dmTFAM-hTFAM and hTFAM-dmTFAM chimeras, respectively. (E,F), PCR genotyping of oTFAM chimeras in which both NTD and CTD support hmtDNA replication (G,H), PCR genotyping of oTFAM chimeras in which only NTD supports hmtDNA replication (I), PCR genotyping of oTFAMs chimeras in which neither NTD, nor CTD supports hmtDNA replication. MTS, matrix targeting sequence of the human ornithine transcarbamylase was appended in front of oTFAM. *, excised by transiently transfecting cells with pMA4854 (Supplementary Figure S1). **, a spurious PCR product in hTFAM-oTFAM PCR due to homology between hTFAM and tdTFAM. mtDNA, nDNA, ΔTFAM, and PhiC31 genotyping as in Figure 1. Subpanel oTFAM-hTFAM, detection of the corresponding chimeras (see diagrams (A,C)). Subpanel hTFAM-oTFAM, detection of the corresponding chimeras (see diagrams (B,D)). Subpanels F + R1 and F + R3, detection of chimera excision (see diagrams (A–D)). See Figure 1A for chimera design and Supplementary Table S1 for primer sequences and predicted amplicon sizes.