Kawarasaki Y, Kasahara S, Kodera N, Shinbata T, Sekiguchi S, Nakano H, Yamane T
Laboratory of Molecular Biotechnology, Division of Molecular and Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Biotechnol Prog. 2000 May-Jun;16(3):517-21. doi: 10.1021/bp000021x.
We prepared a short (29 nucleotides) 5' UTR that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' UTR. The trimmed sequence, designated as TE(37-65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37-65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37-65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37-65) sequence was also performed.
我们制备了一段短的（29个核苷酸）5'非翻译区（UTR），通过对烟草蚀纹病毒5'UTR进行修剪，该序列在小麦胚芽翻译系统中增强了不依赖帽子结构的翻译。修剪后的序列命名为TE(37 - 65)，它取自几种马铃薯Y病毒属病毒的保守区域。携带TE(37 - 65)序列的无帽报告mRNA的翻译效率与有帽的对应物相当，即每1 mL翻译反应混合物可合成5 - 20微克蛋白质。进入TE(37 - 65)序列的核糖体在确定的起始密码子处精确起始多肽合成，这确保了快速且高效的蛋白质截短分析。此外，TE(37 - 65)序列足够短，可以包含在PCR引物中，这提供了一种用于快速基因表达的简单方法，即对目标基因进行PCR扩增，随后进行体外转录和翻译。作为示例，还利用TE(37 - 65)序列对水稻cDNA进行了快速体外表达。
