Plant Virology Laboratory, Plant Protection Institute, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Proc Natl Acad Sci U S A. 1980 Sep;77(9):5302-6. doi: 10.1073/pnas.77.9.5302.
Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC).oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [(32)P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells.
双链 cDNA 已从聚腺苷酸化的马铃薯纺锤块茎类病毒 (PSTV) 模板合成,并通过寡聚 (dC).寡聚 (dG) -加尾程序插入质粒 pBR322 的 Pst I 内切酶位点。四环素抗性氨苄青霉素敏感转化体含有与 PSTV [(32)P]cDNA 互补的序列,一个重组克隆 (pDC-29) 含有 460 个碱基对的插入物。该克隆双链 PSTV cDNA 包含根据 PSTV 已发表的一级序列预测的六个限制内切酶的切割位点,以及 Ava I、Hae III、Hpa II 和 Sma I 每个各一个额外的位点。额外的 Ava I、Hpa II 和 Sma I 位点是由克隆双链 cDNA 中存在第二个 C-C-C-G-G-G 序列解释的。Hae III 消化释放的最大片段约含有 360 个碱基对。这些结果表明我们已经克隆了 PSTV 的几乎整个序列,但克隆的序列与已发表的序列略有不同。源自 pDC-29 插入物的杂交探针允许检测和初步表征与 PSTV 大小相同但极性相反的 RNA 分子。这种 RNA 存在于感染的番茄细胞中 PSTV 的复制过程中。