Isolation of indoleamine oxygenase gene from the mollusc Turbo carnutus and functional convergence

一些古胃足类软体动物，如沟足类和涡轮类，在它们的口腔肿块中含有罕见的~ 40 kDa的肌红蛋白。肌红蛋白可以可逆地结合氧，尽管氧亲和力低于脊椎动物和无脊椎动物的肌红蛋白。氨基酸测序清楚地表明，Sulculus和Turbo肌红蛋白不是由珠蛋白基因进化而来，而是由吲哚胺双加氧酶（IDO）基因进化而来，IDO是色氨酸降解酶。Turbo肌红蛋白基因的结构由14个外显子和13个内含子组成。与已知的ido样肌红蛋白基因相比，除了一个外，所有的剪接都在两个基因之间精确保守。这些肌红蛋白基因的外显子/内含子结构也与人类IDO的（10个外显子-9内含子结构）高度同源：6个内含子的剪接在3个基因之间精确保守，表明这些内含子至少保守了6亿年。将Turbo肌红蛋白cDNA的开放阅读框分别克隆到质粒pQE、pGEX和pET-44中，在大肠杆菌中表达。使用pQE载体不表达肌红蛋白，而大量融合蛋白以不溶性（pGEX）或可溶性（pET-44）形式表达。为了寻找Turbo或Sulculus中可能存在的IDO基因，我们重新详细检查了PCR扩增的基因组DNA片段，发现两个不同的Sulculus片段A和B含有内含子2。片段A与Sulculus肌红蛋白基因完全对应，内含子576 bp。另一方面，含有239 bp内含子的片段B与片段A在核苷酸和翻译氨基酸序列上存在显著差异。详细的序列比较表明，片段B可能来源于推测的Sulculus IDO基因。

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.