Kearney C M, Donson J, Jones G E, Dawson W O
Department of Plant Pathology, University of California, Riverside 92521.
Virology. 1993 Jan;192(1):11-7. doi: 10.1006/viro.1993.1002.
The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.
在构成源自6.4 kb（+）RNA病毒烟草花叶烟草花叶病毒（TMV）的杂交病毒一部分的外源序列中测量突变的积累。所测试的两个外源序列（二氢叶酸还原酶和新霉素磷酸转移酶II）都不是病毒功能上必需的，因此它们应该不受选择压力的影响，并且应该是整个植物中病毒序列漂移的真实度量。四个杂交病毒群体，每个外源序列两个，在本氏烟草的整株植物中传代9 - 10次。通过转化为cDNA、用聚合酶链反应扩增以及对所得细菌克隆进行测序，从这些群体中对序列进行采样。该检测系统的酶所贡献的背景突变率使得能够测量每代每碱基大于10^(-4)个突变的病毒突变率。令人惊讶的是，所有天然和外源基因的突变积累速率都非常低，低于检测程序所能检测到的速率。这种每代每碱基≤10^(-4)个突变的低突变积累速率可能是由于复制酶保真度或群体“瓶颈效应”。序列漂移对于TMV作为载体的大多数用途而言不应是一个实际限制，尽管本研究中观察到的缺失现象可能会带来困难。
