Prapunpoj P, Richardson S J, Fumagalli L, Schreiber G
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.
Mol Biol Evol. 2000 Aug;17(8):1199-209. doi: 10.1093/oxfordjournals.molbev.a026403.
Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.
甲状腺激素参与所有脊椎动物的生长和代谢调节。转甲状腺素蛋白是一种对甲状腺激素具有高亲和力的细胞外蛋白，它决定了这些激素在细胞外区室和细胞内脂质之间的分配。在脊椎动物进化过程中，转甲状腺素蛋白的表达组织模式和基因结构都发生了特征性变化。本研究的目的是确定食虫目动物在哺乳纲一个亚纲——真兽类转甲状腺素蛋白进化中的位置。通过甲状腺素结合和蛋白质免疫印迹分析在成年鼩鼱、刺猬和鼹鼠的血液中鉴定出了转甲状腺素蛋白。转甲状腺素蛋白在肝脏中合成并分泌到血液中，这与其他成年真兽类、鸟类和双门齿有袋类动物的情况相似，但与成年鱼类、两栖类、爬行类、单孔类和澳大利亚多门齿有袋类动物不同。为了表征转甲状腺素蛋白基因的结构和mRNA的加工过程，从刺猬和鼩鼱肝脏的RNA制备了cDNA文库，并分离和测序了全长cDNA克隆。通过聚合酶链反应合成并测序了编码外显子1和2之间剪接位点区域的基因组DNA片段。通过比较基因组和cDNA核苷酸序列推断剪接位置。转甲状腺素蛋白基因在进化过程中的核苷酸序列变化在编码该蛋白N端区域的区域最为明显。发现食虫目动物中转甲状腺素蛋白的推导总体氨基酸序列和N端区域与其他真兽类动物的非常相似，但与有袋类、鸟类、爬行类、两栖类和鱼类中的不同。此外，食虫目动物中转甲状腺素蛋白前体mRNA的剪接模式与其他真兽类动物的比与有袋类、爬行类和鸟类的更相似。因此，与有袋类动物不同，在进化上“较老”的多门齿有袋类动物与进化上“较年轻”的双门齿有袋类动物中转甲状腺素蛋白基因表达模式不同，在真兽类动物中未发现转甲状腺素蛋白进化上的单独谱系。我们得出结论，成年真兽类动物肝脏中转甲状腺素蛋白基因的表达可能在导致现代真兽类物种的谱系分支之前就已出现。
