Characterization of a binding factor that interacts with the sequences upstream of the vaccinia virus thymidine kinase gene.
A small 176 base-pair cloned DNA fragment, representing the nucleotide sequences proximal to the 5'-end of the vaccinia virus thymidine kinase (VV TK) gene, was radiolabeled and used in concert with gel retention assays to detect, partially purify, and characterize a promoter binding factor (PBF) extracted from vaccinia virions. The VV TK PBF was purified from solubilized virus particles by a combination of ion-exchange and DNA-affinity chromatographic procedures. The interaction between VV TK PBF and VV TK promoter sequences was relatively specific in that binding to the radiolabeled probe could be effectively inhibited by unlabeled VV TK promoter or VV TK promoter-specific oligonucleotides, but not by similar-sized fragments of control plasmid DNA. The VV TK PBF did, however, bind to other VV early-promoter elements. Glycerol gradient sedimentation provided an estimate of 130-140 kD for the native molecular weight of VV PBF. This correlated well with data from the purification of VV PBF from radiolabeled VV particles that revealed 2 polypeptides, with molecular weights of 70 and 68 kD that co-purified with VV TK PBF activity. Taken together, these results suggest that a heterodimeric promoter-binding factor, which is present within the cytoplasm of VV-infected cells, is capable of specifically interacting with VV early-promoter elements.
Wilson
E M
EM
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Edbauer
C
C
Hruby
D E
DE
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral; D005814:Genes, Viral; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification; D014616:Vaccinia virus / Q000235:genetics*; D014776:Virus Cultivation
D015342:DNA Probes; D004279:DNA, Viral; D014157:Transcription Factors; D013937:Thymidine Kinase