Polymorphism of thymidylate synthase gene in human gastrointestinal carcinoma cells and its regulational role in protein expression

我们克隆了胸苷合成酶基因，并在5‘非翻译区进行了不同的串联重复序列(2、3、4、5和6个重复)。此外，我们还通过网织红细胞裂解物翻译实验观察了有或没有互补反向序列的各基因型TS mRNA在重复序列上游的翻译活性。包括串联重复序列和互补反向序列的全长TS mRNAs的翻译活性非常低，与重复序列的数量无关。然而，随着重复序列数目的增加，翻译活性明显增加。与全长TS mRNAs相比，无互补反向序列的TS mRNAs的翻译活性显著增加。在没有互补反向序列的TS mRNAs中，随着重复序列数目的增加，翻译活性的提高消失了。有或无互补反向序列的封闭型TS mRNAs的转座活性均增强。网织红细胞裂解产物翻译产物经多克隆TS抗体Western blotting鉴定为TS蛋白。由重复序列和互补反向序列形成的茎环结构应抑制TS mRNA的翻译活性，此外，一些蛋白质可能与茎环结构结合并调节其抑制作用。TS基因5‘端非编码区形成的茎环结构与其结合的蛋白质及其对TS基因翻译活性的影响还有待进一步研究。

We performed cloning of thymidylate synthase (TS) genes with various tandem repeat sequence (2, 3, 4, 5 and 6 repeats) in the 5' untranslated region (5'UTR). Furthermore, we observed translational activity of each genotype TS mRNA with or without complementary reverse sequence in the upstream of the repeat sequence by reticulocyte lysate translation assay. The translational activity of full-length TS mRNAs, which included both of the tandem repeat sequence and complementary reverse sequence, were quirw lowirrespective of the number of the repeat sequence. However, obvious elevation of translational activity was observed when the number of the repeat sequence was increased. Translational activity increased markedly in the TS mRNAs without the complementary reverse sequence compared with the full-length TS mRNAs. The elevation of translational activity with increment of number of the repeat sequence disappeared in the TS mRNAs without the complementary reverse sequence. Capped TS mRNAs, both with and without complementary reverse sequence, showed increase of its trnaslational activity. The products of reticulocyte lysate translation assay were confirmed as TS protein by Western blotting using polyclonal TS anti-body. The stem-loop structure, formed by repear sequence and complementary reverse sequence, should inhibitively control the translational activity of TS mRNA.Further, some protein supposed to bind the stem-loop structure and regulate its inhibitory action. Further studies are required to look for the protein which binds to the stem-loop structure formed in 5'UTR of TS mRNA and its influence on the translational activity of TS mRNA.