Hybridization selection and cell-free translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome

Abstract

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.