The 5′ noncoding region sequence of the Choristoneura biennis entomopoxvirus spheroidin gene functions as an efficient late promoter in the mammalian vaccinia expression system

Abstract

About 100 nucleotides of DNA sequence at the 5′ noncoding region of the Choristoneura biennis entomopoxvirus spheroidin gene was chemically synthesized and inserted into a vaccinia expression vector, interrupting the vaccinia thymidine kinase gene. When the bacterial β-galactosidase gene was introduced downstream of this sequence and a recombinant vaccinia virus containing these inserts was obtained by homologous recombination, β-galactosidase was shown to be expressed at a high level late in the vaccinia infection cycle. The level of β-galactosidase expression was four- to fivefold higher with this spheroidin-vaccinia recombinant virus than with a similar recombinant in which the β-galactosidase gene was under the control of the vaccinia 7.5-kDa promoter. Primer extension and S1 mapping of the 5′ terminus of the β-galactosidase transcript located the transcription initiation site within the spheroidin DNA sequence, confirming the promoter nature of this DNA sequence in the vaccinia system. Dot blot analysis indicated that the difference in β-galactosidase expression with these two recombinant viruses can be attributed to the difference in their transcript levels. We also demonstrated that full promoter activity encoded in the spheroidin 5′ noncoding sequence was contained within a 38-nucleotide DNA fragment.