Schmidt-Puchta W, Dominguez D, Lewetag D, Hohn T
Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland.
Nucleic Acids Res. 1997 Jul 15;25(14):2854-60. doi: 10.1093/nar/25.14.2854.
It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.
有人提出，花椰菜花叶病毒35S RNA及其600个核苷酸长的前导序列采用一种不寻常的翻译过程（翻译跳跃）。利用小麦胚芽体外翻译试验来改进对这一机制的研究。通过缺失、引入稳定的茎环结构以及反义寡核苷酸对基因表达的抑制作用来确定前导序列各部分的作用。结果发现，前导序列的5′端和3′端对于翻译是绝对必需的，而中间部分显然是可有可无的。这些结果证实了原生质体瞬时表达实验已报道的数据。然而，与原生质体实验相反，体外实验数据表明，只需要两端两个相对较短的区域，每个区域大约100个核苷酸。该体外系统为在分子水平上进一步研究跳跃模型以及研究蛋白质在这一机制中的作用提供了工具。还发现水稻东格鲁杆状病毒的前导序列也会发生跳跃。由于小麦既不是花椰菜花叶病毒的寄主植物，也不是水稻东格鲁杆状病毒的寄主植物，这种跳跃似乎不依赖寄主，这一发现与原生质体的早期研究结果不同。
