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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells.
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RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells.

机译：劳斯肉瘤病毒DNA聚合酶指导的RNA定向DNA合成：未感染细胞中4S引物RNA的结构和功能鉴定。

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摘要

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.
机译：劳斯肉瘤病毒的RNA定向DNA聚合酶需要4S RNA分子作为引物，以启动病毒70S RNA基因组上的DNA合成。现在，我们已经根据细胞4S RNA的活性，在体外通过Rous肉瘤病毒的RNA定向DNA聚合酶积极参与DNA合成的启动，从而在功能上确定了未感染细胞中的引物活性。这是通过重建实验完成的，在该实验中，测试了来自未感染禽类细胞的4S RNA将模板活性恢复至已删除所有引物的病毒RNA基因组的能力。使用类似的重建实验来证明鸭，小鼠和人细胞的4S RNA群体中的引物活性。较低的真核或原核细胞似乎没有引物活性。通过直接从大量宿主细胞转移RNA中纯化4S RNA分子并通过二维纸确定该细胞4S RNA种类与Rous肉瘤病毒引物之间的结构相似性，可以对未感染细胞中的Rous肉瘤病毒引物分子进行明确鉴定从RNA物种的T1核糖核酸酶消化物中获得的寡核苷酸的电泳分析。我们得出的结论是，劳斯肉瘤病毒DNA聚合酶可以利用宿主细胞分子作为引物，用于体外启动RNA定向的DNA合成。

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