Wilson E M, Edbauer C, Hruby D E
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Virus Genes. 1988 Oct;2(1):31-48. doi: 10.1007/BF00569735.
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.
一个小的176个碱基对的克隆DNA片段，代表痘苗病毒胸苷激酶（VV TK）基因5'-末端近端的核苷酸序列，经放射性标记后与凝胶滞留试验协同使用，以检测、部分纯化和表征从痘苗病毒粒子中提取的启动子结合因子（PBF）。通过离子交换和DNA亲和层析程序相结合，从溶解的病毒颗粒中纯化出VV TK PBF。VV TK PBF与VV TK启动子序列之间的相互作用具有相对特异性，即与放射性标记探针的结合可被未标记的VV TK启动子或VV TK启动子特异性寡核苷酸有效抑制，但不能被对照质粒DNA的类似大小片段抑制。然而，VV TK PBF确实与其他VV早期启动子元件结合。甘油梯度沉降法估计VV PBF的天然分子量为130 - 140 kD。这与从放射性标记的VV颗粒中纯化VV PBF的数据很好地相关，该数据显示有2种多肽，分子量分别为70和68 kD，与VV TK PBF活性共同纯化。综上所述，这些结果表明，一种存在于VV感染细胞胞质内的异二聚体启动子结合因子能够与VV早期启动子元件特异性相互作用。
