Faras A J, Dibble N A
Proc Natl Acad Sci U S A. 1975 Mar;72(3):859-63. doi: 10.1073/pnas.72.3.859.
The RNA-directed DNA polymerase of Rous sarcoma virus requires a 4S RNA molecule as primer for the initiation of DNA synthesis on the viral 70S RNA genome. We have now functionally identified primer activity in uninfected cells on the basis of the capacity of cellular 4S RNA to actively participate in the initiation of DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus in vitro. This was accomplished by reconstitution experiments in which 4S RNA from uninfected avian cells was tested for its ability to restore template activity to the viral RNA genome from which all primer had been removed. Similar reconstitution experiments were employed to demonstrate a primer activity in the 4S RNA population of duck, mouse, and human cells. Primer activity appears to be absent in lower eukaryotic or prokaryotic cells. Unambiguous identification of the Rous sarcoma virus primer molecule in uninfected cells was accomplished by directly purifying a 4S RNA molecule from the bulk of host cell transfer RNA and establishing structural similarities between this cellular 4S RNA species and the Rous sarcoma virus primer by two-dimensional paper electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the RNA species. We conclude that the Rous sarcoma virus DNA polymerase can utilize a host cell molecule as primer for the initiation of RNA-directed DNA synthesis in vitro.
劳氏肉瘤病毒的RNA指导的DNA聚合酶需要一个4S RNA分子作为引物，以便在病毒70S RNA基因组上起始DNA合成。现在，我们已根据细胞4S RNA在体外积极参与劳氏肉瘤病毒的RNA指导的DNA聚合酶起始DNA合成的能力，在未感染细胞中功能性地鉴定出引物活性。这是通过重组实验完成的，在该实验中，测试了来自未感染禽类细胞的4S RNA恢复已去除所有引物的病毒RNA基因组模板活性的能力。类似的重组实验用于证明鸭、小鼠和人类细胞的4S RNA群体中存在引物活性。在低等真核细胞或原核细胞中似乎不存在引物活性。通过从大量宿主细胞转移RNA中直接纯化一个4S RNA分子，并通过对从该RNA物种的T1核糖核酸酶消化产物获得的寡核苷酸进行二维纸电泳，确定该细胞4S RNA物种与劳氏肉瘤病毒引物之间的结构相似性，从而明确鉴定出未感染细胞中的劳氏肉瘤病毒引物分子。我们得出结论，劳氏肉瘤病毒DNA聚合酶可以利用宿主细胞分子作为引物，在体外起始RNA指导的DNA合成。
