Brini A T, Harel-Bellan A, Korner M, Farrar W L
Laboratory of Molecular Immunoregulation, Frederick Cancer Research Facility, MD 21701-1013.
Biochem Biophys Res Commun. 1989 Apr 14;160(1):268-75. doi: 10.1016/0006-291x(89)91651-3.
We have examined the formation of a DNA "enhancer" - protein complex occurring in situ. Oligonucleotides corresponding to the human immunodeficiency virus (HIV) core enhancer sequence were synthesized, annealed and radiolabeled. The DNA was electroporated either into Jurkat cells or into fresh human peripheral blood T lymphocytes. After the appropriate incubation time and stimulation with various mitogenic agents, cells were lysed and the lysates were electrophoresed on a native polyacrylamide gel. The specific protein-nucleic acid complexes which we obtained were apparently identical to those observed with the "classical" in vitro gel mobility shift assay: one complex seems to be constitutive and the other is induced by mitogens. Additionally competition experiments using "cold" oligonucleotides demonstrated binding specificity in situ. We recommend this novel method for studying DNA-binding proteins and their activation since it requires as few as 10(6) cells, may use primary tissue isolates, and furthermore, allows the rapid assessment of cellular activation signals involved in the post-transcriptional modification of trans-acting factors.
我们检测了原位形成的DNA“增强子”-蛋白质复合物。合成、退火并放射性标记了与人免疫缺陷病毒（HIV）核心增强子序列对应的寡核苷酸。将该DNA通过电穿孔导入Jurkat细胞或新鲜的人外周血T淋巴细胞中。经过适当的孵育时间并用各种促有丝分裂剂刺激后，裂解细胞，裂解物在天然聚丙烯酰胺凝胶上进行电泳。我们获得的特异性蛋白质-核酸复合物显然与“经典”体外凝胶迁移率变动分析中观察到的复合物相同：一种复合物似乎是组成型的，另一种是由促有丝分裂剂诱导的。此外，使用“冷”寡核苷酸的竞争实验证明了原位结合特异性。我们推荐这种研究DNA结合蛋白及其激活的新方法，因为它只需要少至10⁶个细胞，可以使用原代组织分离物，而且还能快速评估参与反式作用因子转录后修饰的细胞激活信号。
