Chimeric Templates and Assays Used to Study Physarum Cotranscriptional Insertional Editing In Vitro

RNAs made in the mitochondrion of Physarum polycephalum are edited relative to their template by the precise addition of nonencoded nucleotides, while they are being synthesized. This insertional editing has been reproduced in vitro during run-on extension of RNAs initiated in vivo, within partially purified mitochondrial transcription elongation complexes (mtTECs), but it does not occur when the mitochondrial polymerase initiates transcription on exogenous cloned DNA. This chapter describes in vitro transcription systems in which mtTEC RNAs are elongated on repositioned parts of the genome or exogenous DNA, in order to investigate how the nontemplated insertions are directed. Restriction enzyme digestion and DNA ligation are used to generate the chimeric templates, and the RNA products are analyzed directly by nuclease dissection (S1 protection followed by RNase T1 digestion) or by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction enzyme analysis or cloning and sequencing.