Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.subsequently added at a concentration of 100 gCi/ml. After incubation at 37 C for 2 to 80 min, the pulses 767 on July 6, 2020 by guest
Concentrated extracts from HeLa cells prepared by the procedure of Sugden and Keller [Sugden, B. & Keller, W. (1973) J. BioL Chem. 248, 3777-3788] can synthesize RNA molecules of discrete lengths in the presence of DNA templates carrying complete viral transcription units. Accurate initiation of transcription was shown to occur at the "major late" promoter and at the promoter for the "early region In" transcription unit of the adenovirus 2 genome. A cloned fragment of adenovirus 2 DNA containing the early region Im transcription unit directs the synthesis ofdiscrete RNA species, many ofwhich correspond in length to spliced early region m mRNAs isolated from adenovirus 2-infected HeLa cells. As shown by hybridization/nuclease SI analysis, RNA splicing and the formation of specific 3' ends are also taldng place in vitro.Since the discovery of
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