Parsons J T, Coffin J M, Haroz R K, Bromley P A, Weissmann C
J Virol. 1973 May;11(5):761-74. doi: 10.1128/JVI.11.5.761-774.1973.
A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.
已开发出一种灵敏且定量的核酸杂交检测方法，用于检测感染鸡细胞中放射性标记的禽肿瘤病毒特异性RNA。在我们的实验中，我们利用了这样一个事实：禽成髓细胞瘤病毒颗粒在放线菌素D存在下合成的DNA（AMV DNA）与劳斯肉瘤病毒施密特-鲁平株（SRV）的70S RNA至少35%的序列互补。将放射性RNA（SRV RNA或从SRV感染的鸡细胞中大量纯化的RNA）与AMV DNA退火，然后进行核糖核酸酶消化和葡聚糖凝胶色谱分析，通过与未标记的70S AMV RNA杂交竞争、在Cs₂SO₄梯度中进行平衡密度梯度离心以及分析其核糖核苷酸组成，得到了被表征为禽肿瘤病毒特异性RNA-DNA杂交体的产物。在用³H-尿苷进行脉冲标记期间合成的病毒RNA量，可以通过在纯化和杂交之前加入由³²P标记的SRV RNA组成的内标来定量。这种定量检测方法被用于确定，在用³H-尿苷标记不同时长的SRV感染鸡细胞中，标记的病毒RNA首先出现在核部分，然后出现在细胞质部分，随后出现在成熟病毒颗粒中。这一观察结果与RNA肿瘤病毒RNA在感染细胞的细胞核中合成的假说一致。
