FIG. 7.

Run-on transcription assay of trnG-UCC transcription in ppr5 mutants. (A) RNAs purified from run-on transcription assays involving plastids isolated from ppr5-1/ppr5-2 seedlings or from their normal siblings (wild-type) were hybridized to the indicated DNA fragments on nylon membranes. Both the trnV-UAC and trnG-UCC loci include a group II intron, and these probes encompass exon and intron sequences. DNA fragments from the mitochondrial (mt) cox1 gene, nuclear (nu) actin, and the pDrive vector into which these probes were cloned were included as controls for nonspecific hybridization. The signals were quantified and used to calculate the mean trnG-to-trnV ratio of two replicate experiments. This was similar for the wild-type (2.6) and ppr5 mutants (1.7). pt, plastid. (B) Run-on transcripts were hybridized to a single-stranded antisense oligonucleotide specific for trnG-UCC exon 2 and to a PCR product spanning the entire trnG-UCC gene. The enhanced signal observed with the oligonucleotide probe relative to the PCR product is typical for experiments of this nature (data not shown) and likely represents the inefficient denaturation of double-stranded DNAs on the filter. WT, wild type.