Alternative mRNA splicing of 3'-terminal exons generates ascorbate peroxidase isoenzymes in spinach (Spinacia oleracea) chloroplasts.
We have isolated two cDNA clones encoding spinach (Spinacia oleracea) stromal and thylakoid-bound ascorbate peroxidase isoenzymes [Ishikawa, Sakai, Yoshimura, Takeda and Shigeoka (1996) FEBS Lett. 384, 289-293]. The gene (ApxII) encoding both chloroplastic ascorbate peroxidase isoenzymes was isolated and the organization of the gene was determined. Alignment between the cDNAs and the gene for chloroplastic ascorbate peroxidase isoenzymes indicates that both enzymes arise from a common pre-mRNA by alternative splicing of two 3'-terminal exons. Genomic Southern-blot analysis supported this finding. The gene spanned nearly 8.5 kbp and contained 13 exons split by 12 introns. The penultimate exon 12 (residues 7376-7530) for the stromal ascorbate peroxidase mRNA consisted of one codon for Asp365 before the TAA termination codon, and the entire 3'-untranslated region, including a potential polyadenylation signal (AATAAA). The final exon 13 (residues 7545-7756) for the thylakoid-bound ascorbate peroxidase mRNA consisted of the corresponding coding sequence of the hydrophobic C-terminal region, the TGA termination codon and the entire 3'-untranslated region, including a potential polyadenylation signal (AATATA). Both exons were interrupted by a 14 bp non-coding sequence. Northern-blot and reverse transcription-PCR analysis showed that the transcripts for stromal and thylakoid-bound ascorbate peroxidase are present in spinach leaves.
Ishikawa
T
T
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631, Japan.
Yoshimura
K
K
Tamoi
M
M
Takeda
T
T
Shigeoka
S
S
D017398:Alternative Splicing / Q000235:genetics*; D060387:Ascorbate Peroxidases; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005091:Exons / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D007438:Introns / Q000235:genetics; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis* / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017421:Sequence Analysis; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D012322:RNA Precursors; D012333:RNA, Messenger; D010544:Peroxidases; D060387:Ascorbate Peroxidases