Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection
Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection
复制标题
DOI：
10.1093/nar/26.10.2426
复制
发表时间：
1998-05-15
影响因子：
14.9
通讯作者：
Simon, AE
中科院分区：
文献类型：
--
作者：
Carpenter, CD;Simon, AE
利用体内遗传选择技术研究了芜菁皱缩病毒（TCV）亚病毒sat-RNA C积累所需的序列和结构。位于sat-RNA C 3 ′端的22个碱基的发夹和6个碱基的单链尾先前被鉴定为负链合成的启动子。22个碱基的区域在所有受感染的植物中积累的sat-RNA中不同，但几乎所有都被预测折叠成发夹结构，其保持6个碱基的尾部作为单链序列。两轮额外的sat-RNA扩增导致四个序列家族“获胜者”，其中三个家族包含多个变体，表明这些序列的进化发生在植物中。四个序列家族获胜者中的三个在茎的基部具有与野生型sat-RNA C相同的3bp。其中两个获胜者共享22个相同碱基中的15个，包括整个茎区和延伸到环中的两个碱基。这些结果通过显示序列和结构都有助于增加sat-RNA C积累的3 ′-末端区域的活性，证明了体内选择方法的实用性。
In vivo genetic selection was used to study the sequences and structures required for accumulation of subviral sat-RNA C associated with turnip crinkle virus (TCV), This technique is advantageous over site-specific mutagenesis by allowing side-by-side selection from numerous sequence possibilities as well as sequence evolution. A 22 base hairpin and 6 base single-stranded tail located at the 3'-terminus of sat-RNA C were previously identified as the promoter for minus strand synthesis, Approximately 50% of plants co-inoculated with TCV and sat-RNA C containing randomized sequence in place of the 22 base hairpin accumulated sat-RNA in uninoculated leaves. The 22 base region differed in sat-RNA accumulating in all infected plants, but nearly all were predicted to fold into a hairpin structure that maintained the 6 base tail as a single-stranded sequence. Two additional rounds of sat-RNA amplification led to four sequence family 'winners', with three families containing multiple variants, indicating that evolution of these sequences was occurring in plants, Three of the four sequence family winners had the same 3 bp at the base of the stem as wild-type sat-RNA C. Two of the winners shared 15 of 22 identical bases, including the entire stem region and extending two bases into the loop. These results demonstrate the utility of the in vivo selection approach by showing that both sequence and structure contribute to a more active 3'-end region for accumulation of sat-RNA C.