Huet J, Phalente L, Buttin G, Sentenac A, Fromageot P
EMBO J. 1982;1(10):1193-8. doi: 10.1002/j.1460-2075.1982.tb00012.x.
Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
制备了针对酿酒酵母天然RNA聚合酶A的小鼠单克隆抗体。通过斑点免疫检测技术筛选后，选择了14个杂交瘤，并在小鼠体内产生抗体。通过印迹免疫检测分析它们的特异性，发现明显偏向于少数RNA聚合酶亚基：A135、A49、A43和A14.5。通过用SDS将RNA聚合酶A解离成其亚基后免疫小鼠，获得了一种针对最大亚基A190的不同单克隆抗体。两种可能识别亚基A135上相同抗原决定簇的抗体抑制了体外RNA合成。用DNA对酶进行预孵育可防止抑制作用，这表明A135亚基在模板结合中起作用。针对A14.5的抗体与酵母核RNA聚合酶的所有三种形式中存在的A14.5 kd亚基相互作用，但不干扰RNA聚合酶活性。这些抗体探针将有助于研究重组转录系统中的亚基功能。
