Max Planck Institut für Biochemie, Abteilung Viroidforschung, D-8033 Planegg-Martinsried, FRG.
EMBO J. 1987 Aug;6(8):2173-83. doi: 10.1002/j.1460-2075.1987.tb02488.x.
A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.
已从悬浮培养的健康马铃薯细胞中分离的核中制备了用于体外加工寡聚类病毒 RNA 的核提取物。借助 SP6 RNA 聚合酶，体外合成了分别含有马铃薯纺锤块茎类病毒（PSTV）的（+）或（-）链的串联单元的线性 RNA 分子，并将其用作加工的底物。当寡聚线性 PSTV（+）RNA 与核提取物孵育时，从它们中准确地切除单体线性分子，并连接到代表类病毒本身的单体 PSTV（+）RNA 环上。寡聚 PSTV（-）RNA 也同样被加工，但效率要低得多。尽管提取物中仍存在其他核酸酶活性，但类病毒加工仍在进行。这些结果证实了我们之前的发现，即在某些内含子和其他 RNA 存在的体外条件下，寡聚 PSTV 不会自动催化加工。这是首次从高等植物中获得体外 RNA 加工系统的报告。
