Figure 3

Analyses of RePro reaction conditions. (A) The 5'-ends of rrn16 transcripts were analyzed by RNase Protection Assay (RPA, lane 2) and Reverse RNase Protection Assay under different conditions (RePro, lanes 3-7). The RePro conditions tested were a full assay (normal, lane 3); addition of sorbitol to a final concentration of 0.33 M (+sorbitol, lane 4); in vitro transcription performed in the presence of [³²P]-UTP but without GTP, CTP, and ATP (-C, G, A, lane 5); broken chloroplasts (broken, lane 6); a control for self-protection of endogenous transcripts without the addition of asRNA (-asRNA, lane 7). (B) The effect of the amount of asRNA in RePro on the signal intensity. 5'-ends of rrn16 transcripts were analyzed by RNase Protection Assay (RPA, lane 2) and Reverse RNase Protection Assay with increasing amounts of unlabeled antisense RNA (RePro): 0.01 μg (lane 3), 0.1 μg (lane 4), 1 μg (lane 5). Mapped rrn16 transcription initiation site (filled circle) is identified by its distance between the transcription initiation site and the first nucleotide of the mature rRNA. The scissors symbol denotes a RNA processing site. Signals of labeled chloroplast transcripts, which are protected against RNase treatment, are marked with asterisks. Note that patterns of additional bands may differ between individual RNA samples. Molecular weight marker in nt is provided on the side (M, lanes 1).