East J L, Knesek J E, Allen P T, Dmochowski L
J Virol. 1973 Nov;12(5):1049-64. doi: 10.1128/JVI.12.5.1049-1064.1973.
The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.
在含有0.008% Triton X-100的情况下，鼠肉瘤病毒内源性指导的RNA依赖性DNA聚合酶反应的DNA产物持续合成长达64小时。更高的去污剂浓度和放线菌素D抑制DNA产物的合成。长期聚合酶反应产生的DNA产物由小的DNA片段组成，碱性蔗糖梯度沉降显示了这一点。通过速率区带离心将酶促DNA产物分离成慢沉降部分和快沉降部分。在含有262 mM NaCl的病毒聚合酶反应中，快沉降DNA是主要部分。通过结合使用S-1核酸酶和胰核糖核酸酶A，测定了慢沉降部分和快沉降部分中单链DNA、双链DNA和DNA-RNA杂交体的量。在70 mM NaCl的标准聚合酶条件下，单链DNA是两个部分中发现的主要DNA形式。相比之下，在262 mM NaCl存在下产生的主要DNA形式是DNA-RNA杂交体。使用S-1核酸酶或胰核糖核酸酶A测量杂交形成的杂交研究不仅表明DNA产物在碱基序列上与RNA基因组互补，而且至少79%至84%的RNA基因组被转录成互补DNA。
