Cox P M, Temperley S M, Kumar H, Goding C R
Marie Curie Research Institute, Oxted, Surrey, UK.
Nucleic Acids Res. 1988 Dec 9;16(23):11047-56. doi: 10.1093/nar/16.23.11047.
The octamer-binding proteins present in HeLa cells, B-cells and malignant melanoma cells were compared by a gel-electrophoresis DNA-binding assay. Using an extract from the malignant melanoma cells a complex was formed using a variety of octamer containing probes that was distinct from those found using either a HeLa or B-cell extract. DNAase 1 footprints and methylation interference patterns of the melanoma-specific octamer-binding protein were indistinguishable from those obtained with the HeLa factor NF-A1, except for preferential binding of the melanoma-specific factor to DNA methylated at two G residues 16 base-pairs 3' to the octamer motif. Competition analyses using a variety of wild-type and mutant probes showed that mutations affecting binding of NF-A1 similarly affected binding of the melanoma octamer-binding factor. These data also revealed the extreme flexibility of the octamer-binding site, with one probe sharing only 4 bases with the 8 base consensus sequence binding efficiently.
通过凝胶电泳DNA结合试验比较了存在于HeLa细胞、B细胞和恶性黑色素瘤细胞中的八聚体结合蛋白。使用恶性黑色素瘤细胞提取物，利用多种含八聚体的探针形成了一种复合物，该复合物与使用HeLa或B细胞提取物所发现的复合物不同。黑色素瘤特异性八聚体结合蛋白的DNA酶1足迹和甲基化干扰模式与用HeLa因子NF-A1获得的模式无法区分，只是黑色素瘤特异性因子优先结合八聚体基序3'端16个碱基对处两个G残基甲基化的DNA。使用多种野生型和突变型探针进行的竞争分析表明，影响NF-A1结合的突变同样影响黑色素瘤八聚体结合因子的结合。这些数据还揭示了八聚体结合位点的极端灵活性，一种探针与8碱基共有序列仅共享4个碱基，却能有效结合。
