Karasev A V, Miroshnichenko N A, Ugarova T Iu
Mol Biol (Mosk). 1989 Jan-Feb;23(1):119-26.
Cell-free translation in Krebs-2 extracts was optimized for RNAs of two plant viruses; potato virus X (PVX, potexvirus), and tobacco mosaic virus (TMV, tobamovirus). PVX and TMV RNAs programmed synthesis of similar sets of polypeptides in both the Krebs-2 extracts and the rabbit reticulocyte lysates, major virus-specific products being the same in molecular weight in both in vitro systems. PVX structural protein (p29) was absent among polypeptides synthesized in the Krebs-2 system but was readily identified by immuno-precipitation among the ones synthesized in the reticulocyte lysate system. The "cap" analog, m7Gpp, inhibited the synthesis of all the polypeptides programmed by PVX RNA in the Krebs-2 system. The synthesis of only a few of the most high molecular weight products in the reticulocyte lysate system was inhibited, the synthesis of a number of low molecular weight products (and among them p29) was even stimulated. Thus, the PVX capped messengers derived from PVX genomic RNA due to its fragmentation with endogenous nuclease activities. The use of the Krebs-2 system allows to avoid activation of internal PVX genes.
针对两种植物病毒的RNA，即马铃薯X病毒（PVX，马铃薯X病毒属）和烟草花叶病毒（TMV，烟草花叶病毒属），对克雷布斯2号提取物中的无细胞翻译进行了优化。PVX和TMV的RNA在克雷布斯2号提取物和兔网织红细胞裂解物中都能指导合成相似的多肽组，两种体外系统中主要的病毒特异性产物分子量相同。在克雷布斯2号系统中合成的多肽中没有PVX结构蛋白（p29），但在网织红细胞裂解物系统中合成的多肽中通过免疫沉淀很容易鉴定出来。“帽”类似物m7Gpp抑制了克雷布斯2号系统中由PVX RNA指导合成的所有多肽。在网织红细胞裂解物系统中，只有少数分子量最高的产物的合成受到抑制，一些低分子量产物（包括p29）的合成甚至受到刺激。因此，由于PVX基因组RNA被内源性核酸酶活性切割，产生了带帽的PVX信使RNA。使用克雷布斯2号系统可以避免激活PVX内部基因。
