DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system

Abstract Homeobox-containing genes encode transcription factors that, via the homeodomain, bind specifically to DNA. To study the DNA-binding properties of the murine homeodomain-containing protein, Hox-2.3, a hybrid expression system was used, combining gene expression by recombinant vaccinia virus (reVV) with bacteriophage T7 transcription. Expression was achieved by co-infecting HeLa cells with two reVVs, one expressing the T7-RNA polymerase-encoding gene directed by the VV promoter, P7.5 , and another containing the Hox-2.3 coding sequence under control of a T7 promoter [Fuerst et al., Mol. Cell. Biol. 7 (1987) 2538–2544]. Co-infected HeLa cells produced large amounts of full-length Hox-2.3 protein. Cytoplasmic and nuclear extracts from these cells were used to examine DNA-binding specificity in vitro. reVV-produced Hox-2.3 protein bound to oligos that contained one or several copies of the common homeodomain-binding site, 5′-TCA-ATTAAAT, and to a lesser extent to multiple (TAA) repeats. Using Southwestern blot analysis, no Hox-2.3-binding sites were detected in a region of the Hox-2 cluster containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes.