Figure 2

A portion of pFOXC1 and pFOXC3 endogenous reverse transcription products is associated with protein. (a) Mitochondrial ribonucleoproteins (mtRNPs) from pFOXC1-containing strains were untreated (lanes 1 and 4), or pretreated with actinomycin D (A; lanes 2 and 5) or RNAse A (R; lanes 3 and 6) for 5 min prior to reverse transcription reactions. Following precipitation, products were heated and electrophoresed in 1.2% agarose gels without (lanes 1-3) or with (lanes 4-6) 0.2% SDS in the gel and loading buffer. (b) Products from endogenous reverse transcription reactions pretreated with actinomycin D and using pFOXC1-containing (lanes 1-6) and pFOXC3-containing (lanes 7-9) mtRNPs were subjected to the following treatments: lane 1, no treatment; lane 2, incubation with proteinase K (K); lane 3, extraction with phenol-CIA (φ); lane 4, incubation with proteinase K followed by extraction with phenol-CIA (K, φ). Lanes 5 and 6 contain acetone-precipitated products recovered from the organic phase of the phenol extractions shown in lanes 3 and 4, respectively. Minus-strand cDNA products from pFOXC3-containing mtRNPs were subjected to the following treatments: lane 7, no treatment; lane 8, incubation with proteinase K followed by extraction with phenol-CIA (K, φ); lane 9, extraction with phenol-CIA (φ). Products were heated at 65°C in 0.2% SDS followed by electrophoresis in a 1.2% agarose gel containing 0.2% SDS. Marker sizes from 5'-end labeled λ-Pst I restriction fragments are indicated in kb pairs on the left. The location of the wells is indicated with a gray arrow. The full-length (-) strand cDNA product is indicated on the right with a black arrow and a high molecular weight band detected in reactions lacking actinomycin D is indicated with an asterisk.