Figure 3

Protein-primed reverse transcription and identification of a nucleotide-linked protein in exogenous reverse transcription reactions. Mitochondrial ribonucleoprotein (mtRNP) particles were treated with micrococcal nuclease (MN), incubated with actinomycin D, and used in reactions having [α-³²P]dATP with various combinations of deoxyribonucleotide triphosphates (dNTPs), either in the absence or presence of an exogenous RNA, as indicated. (a) MN-treated pFOXC1-containing mtRNP particles were incubated in the absence (lane 1) or presence of a 92 nucleotide RNA corresponding to the 3' terminus of the pFOXC1 transcript with a full complement of nucleotides (lane 2). Reactions in lanes 3-5 were performed as in lane 2, but a single unlabeled nucleotide was omitted, as indicated. Products from exogenous reactions having a full complement of nucleotides and exogenous RNA were post-treated with proteinase K (K; lane 6), or extracted with phenol-CIA (φ; lane 7), prior to precipitation with ethanol. Products from a duplicate reaction of that in lane 2 were recovered from the aqueous (lane 8) and organic (lane 9) phase. (b) MN-treated pFOXC3-containing mtRNPs incubated in the absence (lanes 2-4) or presence (lanes 1, 5 and 6) of a 98 nucleotide RNA corresponding to the 3' terminus of the pFOXC3 transcript with [α-³²P]dATP and combinations of unlabeled nucleotides, as indicated. Products from a duplicate reaction of that shown in lane 5 were post-treated with proteinase K (K; lane 6). All reactions were boiled in Laemmli buffer prior to separation via 4-20% gradient SDS-PAGE. Prestained protein size markers are indicated on the left in kDa. Protein-linked products and (-) strand cDNA products are indicated on the right.