Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene

Abstract

The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.