DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system.
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.
de Jong
R
R
Hubrecht Laboratory, Utrecht, The Netherlands.
de Laaf
L
L
Vennema
H
H
Meijlink
F
F
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005801:Genes, Homeobox / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013604:T-Phages / Q000378:metabolism; D014616:Vaccinia virus / Q000378:metabolism
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins