VAI Gene Research Articles

In vitro accurate initiation of transcription on the adenovirus type 2 IVa2 gene which does not contain a TATA box.

In vitro initiation of transcription has been studied on the IVa2 gene of the adenovirus type 2 ( Ad2 ) which is expressed at intermediate time after infection. This gene does not contain a TATA box around 30 bp upstream from the cap site. By analyses of the in vitro runoff products by size and by single strand nuclease protection mapping, it was concluded that the in vitro initiation occurred accurately at two in vivo cap sites with low but detectable efficiency. By using cloned IVa2 DNA, which is free from the major late promoter, it was suggested that the low efficiency of the in vitro initiation on the IVa2 gene may not be due to close location of the two genes but may rather reflect the efficiency of the promoter itself. The cell-free extracts from both uninfected and infected HeLa cells supported the accurate initiation of transcription in vitro with almost the same efficiency. In addition, the cell free extract directed an artificial initiation of transcription in vitro by recognizing a TATA box located 140 bp upstream from the cap sites.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Transcriptional control regions of the adenovirus VAI RNA gene

By constructing deletion mutations in cloned adenovirus types 2 and 5 VAI genes and measuring the ability of altered templates to direct transcription of VAI RNA in HeLa cell extracts, we have located two transcriptional control regions. The first is an intragenic region located between positions +9 and +72 relative to the 5′ end of the VAI(A) RNA. Those deletions examined within these sequences abolished the transcription of mutant templates in HeLa cell extracts. The second control region includes 5′ flanking sequences which abut the VAI coding region. Mutations here can reduce the efficiency with which the VAI gene is transcribed. Nucleotide sequence similarities were noted on comparison of the VAI intragenic control region to tRNA sequences, which lead us to speculate that the transcriptional regulation of these two types of genes may be quite similar; the adenovirus VA genes may even have evolved from a tRNA gene(s).

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.