Transcription of adenoviral genetic information in isolated nuclei. Characterization of viral RNA sequences synthesized in vitro.

The virus-specific RNA sequences synthesized in nuclei isolated from adenovirus type 2-infected HeLa cells comprise a fraction of the total RNA similar to that observed with RNA made in vivo. By 16 h after infection, for example, some 25% of the RNA made in isolated nuclei is transcribed from adenoviral DNA. Only 10 to 15% of the adenoviral RNA sequences synthesized in nuclei isolated during the late phase of infection are transcribed by form III RNA polymerase. This RNA, whose synthesis is resistant to 0.5 microgram/ml, but sensitive to 200 microgram/ml of alpha-amanitin, sediments at about 5 S in denaturing sucrose gradients and must, therefore, represent the virus-associated RNAs. The remainder of the sequences are transcribed by form II RNA polymerase and sediment as several RNA species in denaturing sucrose gradients. The largest of these exhibits the size, 55 to 60 S, expected of a complete transcript of the major, adenoviral transcriptional unit expressed during the late phase. Hybridization of RNA 32P-labeled in nuclei isolated during the late phase of infection to restriction endonuclease fragments of adenoviral DNA immobilized on nitrocellulose filters suggests that sequences of this transcriptional unit are indeed transcribed in vitro. To make a detailed assessment of the fidelity of transcription in isolated nuclei, transcription reactions were performed in the presence of 5-mercuricytidine 5'-triphosphate and the RNA mercurated in vitro separated from endogenous RNA by chromatography on sulfhydryl-agarose columns by a stringent procedure. After demercuration, RNA made in nuclei isolated 20 h following adenovirus type 2 infection was hybridized to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus type 2 DNA. Such RNA is complementary to the r strand of adenoviral DNA from 16.6 units to a point to the right of 98.3 units. These sequences comprise the major transcriptional unit expressed during the late phase (Fig. 1). It is therefore clear that the fidelity of transcription of adenoviral DNA by form II RNA polymerase is preserved in isolated nuclei. Two 1-strand transcriptional units, those of the IVa2 and ts36 genes (see Fig. 1) are also active at 20 h after infection. The results of similar analysis of RNA made in nuclei isolated 4 and 12 h after infection are also presented and discussed in terms of the mapping of individual transcriptional units within the type 2 adenoviral genome and the temporal regulation of adenoviral gene expression during productive infection.