Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment.
Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment.
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来自 PCR 扩增的蚕豆斑驳溴病毒 cDNA 克隆的感染性转录本和 RNA 3 片段中前导区的可变性质。
DOI：
10.1099/0022-1317-75-3-693
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Bujarski,JJ
中科院分区：
文献类型：
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作者：
Pogany,J;Huang,Q;Romero,J;Nagy,PD;Bujarski,JJ
蚕豆斑驳病毒 (BBMV) 的基因组包含三个正义 ssRNA 片段，每个片段均以 m7GpppA 封端。采用逆转录酶 PCR (RT-PCR) 和高保真 Vent DNA 聚合酶构建了 4 种 BBMY 菌株的全长可转录 cDNA 克隆。转录的 BBMY RNA 含有 5' 非病毒 G 残基，虽然延迟，但产生的症状与在感染真正病毒粒子 RNA 的植物中观察到的症状相似。转录本在原生质体中复制效率低下。相比之下，转录衍生的后代 BBMV RNA 具有修复的末端，与真正的 BBMV RNA 一样具有传染性，并且在原生质体中复制到高水平。合成 RNA 的体外翻译证实了先前提出的 BBMV 基因表达策略。对 Bawden 毒株的病毒体 RNA 进行测序，发现了两种形式的 BBMV RNA 3 成分，较长的形式含有 21 个 5' 额外核苷酸，这些核苷酸是由两个短 5' 前导区重复衍生而来。两种RNA 3 形式的相对浓度被发现具有宿主依赖性，较长的形式在蚕豆和克利夫兰烟草感染中占主导地位，而较短的形式在豆类感染中占主导地位。
The genome of broad bean mottle bromovirus (BBMV) contains three positive-sense ssRNA segments, each capped with m7GpppA. Full-length transcribable cDNA clones for four strains of BBMY were constructed by employing reverse transcriptase-PCR (RT-PCR) and a high fidelity Vent DNA polymerase. The transcribed BBMY RNAs contained a 5′ non-viral G residue and, although delayed, produced symptoms similar to those observed in plants infected with authentic virion RNAs. The transcripts replicated inefficiently in protoplasts. In contrast, transcript-derived progeny BBMV RNAs had the repaired termini, were as infectious as the authentic BBMV RNAs and replicated to high levels in protoplasts.In vitrotranslation of synthetic RNAs confirmed the previously proposed gene expression strategy for BBMV. Sequencing of virion RNAs from the Bawden strain revealed two forms of BBMV RNA 3 components, the longer form containing 21 5′ extra nucleotides derived by the duplication of two short 5′ leader regions. The relative concentration of the two RNA 3 forms was found to be host-dependent, with the longer form prevailing in broad bean andNicotiana clevelandiiinfections and the shorter form in bean infections.