CDNA CLONING OF THE COMPLETE GENOME OF TOBACCO MOSAIC-VIRUS AND PRODUCTION OF INFECTIOUS TRANSCRIPTS
CDNA CLONING OF THE COMPLETE GENOME OF TOBACCO MOSAIC-VIRUS AND PRODUCTION OF INFECTIOUS TRANSCRIPTS
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DOI：
10.1073/pnas.83.6.1832
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发表时间：
1986-03-01
影响因子：
11.1
通讯作者：
GRANTHAM, GL
中科院分区：
文献类型：
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作者：
DAWSON, WO;BECK, DL;GRANTHAM, GL
将烟草花叶病毒（TMV）全基因组克隆成一系列亚基因组cDNA克隆。分别克隆了TMV 5”和3”末端的cDNA序列。使用合成寡核苷酸引物在5“末端产生Pst I位点，而使用不同引物在3”末端产生Nde I位点。该策略允许通过在限制性内切酶切割后用核酸外切酶VII处理从克隆的cDNA插入片段中去除非TMV序列。将Pst I接头添加到TMV 3“末端cDNA。将亚基因组cDNA片段连接在一起形成几个独立的全基因组结构，通过Pst I消化，可以将TMV cDNA序列作为单个片段干净地切除。将全基因组TMV cDNA立即连接到该质粒的下游。噬菌体启动子pPM 1和体外转录大肠杆菌RNA聚合酶。来自四个全基因组cDNA构建体中的三个的RNA转录物是感染性的，即使它们在3“末端含有6个非TMV核苷酸。来自在5“末端具有6个额外核苷酸的构建体的转录物也具有感染性。来自感染cDNA转录物的植物的子代病毒似乎与亲本病毒相同。基因组相同区域的独立cDNA克隆的限制性内切酶图谱彼此相同，与TMV报道的核苷酸序列一致。另外，4个独立的cDNA克隆的5“末端附近的200个核苷酸的序列彼此相同并且与公开的序列相同，这表明TMV的独立分离物可能具有显著相似的序列。
The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5'' and 3'' ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5'' terminus, whereas a different primer was used to generate an Nde I site at the 3'' terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage. Pst I linkers were added to TMV 3'' terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent full-genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion. Full-genomic TMV cDNA was ligated immediately down-stream from the .lambda. phage promotor from pPM1 and transcribed in vitro with Escherichia coli RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3'' end. Transcripts from a construction with 6 extra nucleotides at the 5'' end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5'' termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.