A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) W HindIII D DNA fragment by marker rescue of a DNA-temperature-sensitive mutant, tsU7, using cloned fragments of the viral genome. The region of W DNA containing the tsl7 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that

more »

... tion from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb Bglll fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the tsU7 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the tsl7-encoded polypeptide was not identified, it was noted that in tsl7-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the W replicative cycle.