| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Some members of archaeogastropodic molluscs such as Sulculus and Turbo contain unusual 〜40 kDa myoglobin in their buccal masses. The myoglobin can bind oxygen reversibly, although the oxygen affinity is lower than those of vertebrate and invertebrate myoglobins. The amino acid sequencing clearly showed that Sulculus and Turbo myoglobins evolved not from globin gene but from gene for indoleamine dioxygenase (IDO), a tryptophan-degrading enzyme. The structure of Turbo myoglobin gene has been determined to consist of 14 exons and 13 introns. Compared with the known gene for Sulculus IDO-like myoglobin, all splice junctions but one are conserved exactly between two genes. The exon/intron organization of these myoglobin genes is also highly homologous with that (10 exon-9 intron structure) of human IDO : splice junctions of 6 introns were exactly conserved between three genes, suggesting that these introns have been conserved for at least 600 million years. The open reading frame of Turbo myoglobin cDNA was cloned into the plasmids pQE, pGEX and pET-44, and expressed in E. coli. No myoglobin was expressed in use of pQE vector, while a large amount of fusion protein was expressed as insoluble (pGEX) or soluble (pET-44) forms. To look for putative IDO gene in Turbo or Sulculus, we reexamined the genomic DNA fragments amplified by PCR in full detail, and found two distinct Sulculus fragments A and B containing intron 2. The fragment A corresponded exactly to the myoglobin gene of Sulculus, containing 576 bp intron. On the other hand, fragment B, containing 239 bp intron, differed significantly from fragment A in nucleotide and translated amino acid sequences. Detailed sequence comparison suggests that fragment B may be derived from putative IDO gene of Sulculus.
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