Masuta C, Kuwata S, Takanami Y
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa.
J Biochem. 1988 Nov;104(5):841-6. doi: 10.1093/oxfordjournals.jbchem.a122560.
Full-length cDNA of a satellite RNA (Strain Y) which induces bright yellow symptoms on tobacco plants infected with cucumber mosaic virus (CMV) was cloned and sequenced. The published sequence of the satellite RNA was revised with three possible differences (residues 161, 167, and 173) and a nucleotide insertion at residue 234. The satellite cDNA was then inserted into a commercially available transcription vector. In vitro transcription products from the recombinant plasmid harbored 24 non-viral bases at their 5' ends and had very low infectivity when coinoculated with CMV. After removal of the extra 5' sequence of the transcripts with RNase H, the infectivity of the transcripts increased markedly. Analysis of the effects of extra 5' sequences of several lengths confirmed the importance of natural 5' ends for biological activity of the satellite. Trimming down to 6-9 extra bases at the 5' end enhanced the infectivity of the transcripts by 10-fold, although the specific activity of the natural satellite is still 100-fold higher. Dideoxynucleotide sequence analysis proved that the progeny satellite RNA did not retain the 24 non-viral bases at the 5' end of the transcript from pIBI 31-MC.
克隆并测序了一种卫星RNA（Y株系）的全长cDNA，该卫星RNA可在感染黄瓜花叶病毒（CMV）的烟草植株上诱发亮黄色症状。已发表的卫星RNA序列有三处可能的差异（第161、167和173位残基）以及第234位残基处的一个核苷酸插入，据此对该序列进行了修订。随后将卫星cDNA插入一种市售转录载体。重组质粒的体外转录产物在其5'端含有24个非病毒碱基，与CMV共接种时感染性很低。用核糖核酸酶H去除转录本多余的5'序列后，转录本的感染性显著提高。对几种长度的多余5'序列的影响进行分析，证实了天然5'端对卫星生物活性的重要性。将5'端修剪至6 - 9个额外碱基可使转录本的感染性提高10倍，不过天然卫星的比活性仍高出100倍。双脱氧核苷酸序列分析证明，子代卫星RNA并未保留来自pIBI 31 - MC转录本5'端的24个非病毒碱基。
