Lucas J J, Ginsberg H S
J Virol. 1971 Aug;8(2):203-14. doi: 10.1128/JVI.8.2.203-214.1971.
By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 mum 5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of (3)H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the (3)H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition ("presaturation" hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4S. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication-it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.
通过使用脱氧核糖核酸（DNA）-核糖核酸（RNA）杂交技术，在存在2 μmol 5-氟脱氧尿苷的情况下，于感染2型腺病毒的细胞的总RNA制剂中，感染后6小时检测到病毒特异性RNA（cRNA）。在不存在5-氟脱氧尿苷的情况下，（3）H-尿苷掺入病毒cRNA的量持续增加，直至感染后20小时，此时进入RNA的（3）H-尿苷中约40%存在于病毒特异性RNA中。当从感染细胞的细胞质提取物获得的多核糖体组分制备RNA时，感染后3小时（即病毒DNA合成开始前3至4小时）检测到病毒导向的转录。通过DNA-RNA杂交抑制（“预饱和”杂交竞争）技术比较了感染后不同时间合成的病毒cRNA种类。比较了三种杂交抑制技术。这些技术在第一次杂交反应后分离DNA-RNA复合物的方式上有所不同。根据所采用的程序，测量到不同程度的抑制。如果在杂交前将抑制性RNA种类碱降解至约4S的均匀大小，则可以基本消除这种变化。如果在与标记RNA进行第二次杂交之前，通过使用涉及严格洗涤（最好包括核糖核酸酶处理）的程序分离DNA-RNA复合物，则可以使用未降解的RNA。当使用严格的杂交抑制程序时，可以区分出三类病毒特异性RNA种类：（i）早期RNA I类，其合成在病毒DNA复制之前开始，并在病毒DNA复制开始后的某个时间停止——它约占早期RNA种类的70%，感染后18小时明显降解；（ii）早期RNA II类，其合成在病毒DNA复制之前开始，并且在感染后期显然以更高的速率继续；以及（iii）晚期RNA，其合成在病毒DNA合成开始之后开始。
