Characterization of the mitochondrial orfB gene and its derivative, orf224, a chimeric open reading frame specific to one mitochondrial genome of the "Polima" male-sterile cytoplasm in rapeseed (Brassica napus L.).

orf224 is a novel reading frame present upstream of the atp6 gene in the mitochondria of "Polima" cms cytoplasm of rapeseed. In order to determine the origin of orf224, the sequences homologous to orf224 were isolated and characterized. Sequence analysis indicated that orf224 originated by recombination events involving the 5'-flanking region and the amino-terminal segment of the coding region of orf158 (well-known as orfB in other plants), part of exon 1 of the ribosomal protein S3 (rps3) gene, and an unidentified sequence. Transcripts of the orf158 gene were found to be edited at three positions, one of which induces an amino-acid change, while orf224 transcripts have only one RNA editing site within the region homologous to the rps3 gene. This editing site is also present in the proper rps3 transcripts. This result indicates that editing of orf224 occurred because of the sequence homology to rps3. Polyclonal antibodies prepared against a rapeseed ORF158 fusion protein specifically recognize a 18-kDa protein in the membrane fractions of mitochondria from both normal and cms rapeseed.