The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants.Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from theyJ7 gene (ORE31), which is conserved in the chloroplast genomes of many other plant species.RNA editing is one of the posttranscriptional events that has been found to occur in a wide range of organisms (1). It leads to the formation of functional RNAs through alterations of nucleotide sequences of primary transcripts by either base substitutions or nucleotide insertions and deletions. In plants, RNA editing was first reported in the mitochondria of wheat and Oenothera (2-4). To date, several hundred RNA editing sites, mostly C-to-U and, rarely, U-to-C conversions, have been found in the mitochondria of maize, wheat, Oenothera, and other plant species (5). Subsequently, the occurrence of RNA editing in chloroplasts was reported for the first time in maize rpl2 transcript (6). Later, several instances of RNA editing were found in angiosperm chloroplasts, but the number of editing sites remains limited (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) (21). Based on the alignment of predicted amino acid sequences, many potential RNA editing sites were found in the protein-coding regions of the transcripts from black pine chloroplast DNA. We examined such potential sites and identified 26 edited sites in mRNAs from 12 genes/ORFs. Interestingly, we also found two C-to-U editing events that created an initiation and a stop codon within a single transcript resulted in the formation of a new polypeptide-coding frame at the RNA level. MATERIALS AND METHODSTwo-week-old Pinus thunbergii seedlings were supplied from Oji Institute for Forest Tree Improvement. The leaves and stems were frozen in liquid nitrogen, and total RNA and DNA were prepared as described (22). cDNA synthesis was carried out by using cDNA cycle kit (Invitrogen) using random primers and 1 ,ug of DNase-treated total RNA as template. Search for potential editing sites was carried out using a HP9000-845 computer (Hewlett-Packard). cDNA and chloroplast genomic DNA were amplified by polymera...

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