Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.
Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.
复制标题
啮齿动物未成熟 T 细胞系中 SV40 增强子基序的活性和白细胞介素 1 反应性。
作者：
E. Espel;C. Fromental;P. Reichenbach;M. Nabholz
我们在未成熟的啮齿动物T细胞系PC60中分析了SV40增强子的活性和单个基元的白细胞介素1 （il - 1）响应性。瞬时转染实验表明，GT - I+ GT - IIC基序、TC - II或P基序的四聚体在PC60中具有显著的增强子活性，而Octamer和SphI+II基序对启动子强度都没有可检测到的影响。两个基序，TC - II和P，对il - 1的刺激有强烈反应。DNase I和核提取物的甲基化保护实验显示SV40增强子的TC - II区域有特定的足迹。PC60细胞暴露于il - 1会增加其强度。TC‐II序列形成了几个在带移实验中检测到的复合物。所涉及的分子都具有与NF - kappa b相似的序列特异性，令人惊讶的是，对照或il - 1处理的细胞提取物的条带移位仅略有不同。然而，如果GTP加入到结合反应中，对照细胞提取物形成的条带强度会大大降低，而il - 1处理细胞提取物形成的条带会与来自前B细胞系的NF - kappa B共同迁移。结果表明，在PC60中，IL1通过激活已经存在于细胞核中的分子来诱导NF - κ B活性。
We have analysed the enhancer activity and the interleukin 1 (IL1) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT‐I plus GT‐IIC motifs, the TC‐II or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC‐II and P, strongly respond to stimulation by IL1. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC‐II region of the SV40 enhancer. Exposure of PC60 cells to IL1 increases their intensity. The TC‐II sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF‐kappa B. Surprisingly, band shifts with extracts from control or IL1 treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from IL1 treated cells form a single retarded complex that co‐migrates with NF‐kappa B from a pre‐B cell line. The results suggest that in PC60 IL1 induces NF‐kappa B activity by activating molecules that are already in the nucleus.