Craig E A, Raskas H J
J Virol. 1974 Oct;14(4):751-7. doi: 10.1128/JVI.14.4.751-757.1974.
The RNA sequences and RNA size classes transcribed early in productive infection with adenovirus 2 were analyzed by RNA-DNA hybridization. Two independent procedures demonstrated that early cytoplasmic viral RNA is composed of two sequence classes, class I which is absent or present in greatly reduced quantities at 18 h, and class II which persists throughout the infection. When the sequences in early viral RNA were analyzed by hybridization-inhibition studies, the hybridization of early [(3)H]RNA was inhibited only 50% by RNA from cultures harvested late (18 h) in infection. Liquid hybridizations with radioactive viral DNA confirmed that early RNA includes two classes. Duplex formation of RNA with (32)P-labeled viral DNA was assayed by hydroxylapatite chromatography and resistance to S(1) nuclease digestion. Both methods showed that the cytoplasmic RNA present early in infection annealed 12 to 15% of the viral DNA; late cytoplasmic RNA hybridized 21 to 25% of the DNA. Mixtures of early plus late cytoplasmic RNAs hybridized 30 to 34% of the viral DNA, demonstrating the reduced concentration of early class I RNA in the late RNA preparations. Experiments were performed to correlate class I and class II early RNA with size-fractionated cytoplasmic RNA synthesized early in infection. Fractionation of RNA by gel electrophoresis or sucrose gradient centrifugation confirmed three major size classes, 12 to 15S, 19 to 20S, and 26S. Total cytoplasmic RNA and RNA selected on the basis of poly(A) content contained the same size classes of viral RNA. In standard electrophoresis conditions, the 19 to 20S viral RNA could be resolved into two size classes, and the distribution of 12 to 15S RNA also indicated the presence of more than one size component. Hybridization-inhibition studies under nonsaturating conditions were performed with 26S, 19 to 20S, and 12 to 15S viral RNAs fractionated by gel electrophoresis. Late RNA inhibited the hybridization of 26S RNA only 20%, 19 to 20S RNA was inhibited 45%, and 12 to 15S RNA was inhibited 50%. When 18 to 19S and 12 to 15S viral RNAs purified by sucrose gradient centrifugation were similarly analyzed, late RNA inhibited hybridization of 18 to 19S RNA 50%, and the annealing of 12 to 15S RNA was inhibited 70%.
用RNA-DNA杂交分析法对腺病毒2有效感染早期转录的RNA序列和RNA大小类别进行了分析。两项独立的实验程序表明，早期细胞质病毒RNA由两类序列组成，I类在感染18小时时不存在或含量大幅降低，II类在整个感染过程中持续存在。当通过杂交抑制研究分析早期病毒RNA中的序列时，来自感染后期（18小时）收获的培养物的RNA仅抑制早期[（3）H]RNA杂交的50%。用放射性病毒DNA进行的液相杂交证实早期RNA包括两类。通过羟基磷灰石色谱法和对S（1）核酸酶消化的抗性来测定RNA与（32）P标记的病毒DNA的双链体形成。两种方法均表明，感染早期存在的细胞质RNA与12%至15%的病毒DNA退火；后期细胞质RNA与21%至25%的DNA杂交。早期和后期细胞质RNA的混合物与30%至34%的病毒DNA杂交，这表明后期RNA制剂中早期I类RNA的浓度降低。进行了实验以将早期I类和II类RNA与感染早期合成的按大小分级的细胞质RNA相关联。通过凝胶电泳或蔗糖梯度离心对RNA进行分级分离，证实了三个主要大小类别，即12至15S、19至20S和26S。总细胞质RNA和根据poly（A）含量选择的RNA含有相同大小类别的病毒RNA。在标准电泳条件下，19至20S病毒RNA可分为两个大小类别，12至15S RNA的分布也表明存在不止一种大小成分。用凝胶电泳分离的26S、19至20S和12至15S病毒RNA在非饱和条件下进行杂交抑制研究。后期RNA仅抑制26S RNA杂交的20%，19至20S RNA被抑制45%，12至15S RNA被抑制50%。当对通过蔗糖梯度离心纯化的18至19S和12至15S病毒RNA进行类似分析时，后期RNA抑制18至19S RNA杂交的50%，12至15S RNA的退火被抑制70%。
