An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells.
An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells.
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通过定点诱变衍生的琥珀抑制 tRNA 基因：哺乳动物细胞中的克隆和功能。
DOI：
10.1073/pnas.79.19.5813
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发表时间：
1982
影响因子：
11.1
通讯作者：
Sharp,PA
中科院分区：
文献类型：
--
作者：
Laski,FA;Belagaje,R;RajBhandary,UL;Sharp,PA
我们描述的合成，克隆，表达，并在哺乳动物细胞抑制tRNA基因的体内功能。通过对克隆到重组单链噬菌体M13mp5中的非洲爪蟾酪氨酸tRNA基因进行“引物定向诱变”，我们获得了一个琥珀色抑制tRNA基因，该基因在反密码子序列中具有核苷酸变化——GTA通向CTA。以猴病毒40 (SV40) DNA为载体（SV40- trnatyrsu +），将抑制因子（Su） tRNA基因导入猴肾细胞（CV-1）。用含有突变体而非野生型tRNA基因的病毒感染CV-1细胞，通过抑制血清型2-SV40杂交腺病毒的琥珀色突变，可以产生功能性的琥珀色抑制因子tRNA。通过从SV40-tRNATyr （Su+）重组病毒感染的CV-1细胞中分离总tRNA，并将其用于证明烟草花叶病毒RNA在网状细胞提取物中体外翻译时琥珀密码子的通读，进一步证明了这些琥珀色突变是tRNA介导的。有趣的是，在CV-1细胞中琥珀色抑制基因的扩增并不会干扰SV40的产生，这表明琥珀色密码子的抑制可能不会对哺乳动物细胞代谢造成很大的危害。
We describe the synthesis, cloning, expression, and in vivo function of a suppressor tRNA gene in mammalian cells. By using "primer-directed mutagenesis" on a Xenopus laevis tyrosine tRNA gene cloned into the recombinant single-strand phage M13mp5, we have generated an amber suppressor tRNA gene that has a nucleotide change--GTA leads to CTA--in the anticodon sequence. The suppressor (Su) tRNA gene was introduced into monkey kidney cells (CV-1) by using simian virus 40 (SV40) DNA as vector (SV40-tRNATyrSu+). CV-1 cells infected with virus containing the mutant, but not the wild-type, tRNA gene produce a functional amber suppressor tRNA as indicated by suppression of amber mutations in co-infecting adenovirus serotype 2-SV40 hybrids. Further evidence that suppression of these amber mutations is tRNA mediated was derived by isolation of total tRNA from CV-1 cells infected with the SV40-tRNATyr (Su+) recombinant and its use in demonstration of read through of an amber codon during in vitro translation of tobacco mosaic virus RNA in reticulocyte extracts. Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.