Complete Nucleotide Sequences of Two Adjacent Early Vaccinia Virus Genes Located Within the Inverted Terminal Repetition

Abstract

The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65 :499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine · thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3′ end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3′ ends of the two transcripts were located within 100 bp of each other. Although there are adenine · thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of repeated sequences downstream of T clusters

“…The sequence extending from the 5' end to the third 3' end in Fig. 3 was identical to that previously obtained by analysis of cloned genomic DNA (26). The absence of splicing indicates that the loss of predicted N-terminal and C-terminal sequences in mature VGF (23) results from processing at the protein level.…”

“…There are about 100 early genes dispersed through the vaccinia virus genome, several of which have been' transcriptionally mapped and sequenced (7,10,12,26,27). Attention has been directed primarily to the RNA start sites and the promoter sequences present upstream.…”

“…We chose to analyze cDNA copies of mRNA that encodes the vaccinia virus growth factor (VGF). This gene is located within the inverted terminal repetition and was transcriptionally mapped and sequenced several years ago (26). Recently, it was found to be structurally (3,6,20) and functionally (14,23,24) related to epidermal and transforming growth factors of higher eucaryotes.…”

“…The library was also screened with probes for some previously sequenced early vaccinia virus genes. Of 2,500 colonies, 16 were positive when probed with a 250-base-pair (bp) AccI-DdeI DNA segment containing the 3' terminus of the VGF gene (26), whereas only 1 was positive when probed with the thymidine kinase gene. The low number of thymidine kinase cDNAs is in agreement with data suggesting that this gene has a relatively weak promoter (1,16).…”

Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase

“…The use of monoclonal antibodies in conjunction with poxvirus DNA libraries contained within a suitable expression vector such as Xgtll (39) would allow rapid localization of genes for individual proteins within the viral genome. It should be noted that this approach is particularly suitable for poxviruses because to date, poxvirus genes appear to lack introns (15,(34)(35)(36)(37)). An expression vector-poxvirus DNA genome library can, therefore, be prepared and used directly for gene mapping without having to first prepare cDNA copies of the viral transcripts.…”