Kawakami K, Watanabe Y, Araki M, Nagano K
Department of Biology, Jichi Medical School, Tochigi, Japan.
J Neurosci Res. 1993 Jun 1;35(2):138-46. doi: 10.1002/jnr.490350204.
We analyzed cis-acting elements regulating the expression of the gene encoding adhesion molecule on glia (AMOG) in primary cultured astrocytes from newborn rat cerebrum and cerebellum. The relative promoter activities among the series of 5' sequential deletion mutants are similar to those observed in B103 (rat neuroblastoma cell line) cells. The previously identified AMRE (AMOG regulatory element) of the GAGGCGGGG sequence functions as a positive regulatory element, not only in B103 cells, but also in astrocytes. Binding factors to the element were identified as Sp1 based on the following observations using nuclear extracts from the astrocytes and B103 cells: (1) The interaction of the factors with AMRE analyzed by DNase I footprinting and methylation interference analyses was similar to that of Sp1; (2) The binding of the factors to AMRE competed with an oligonucleotide containing the authentic Sp1 consensus sequence; (3) Sp1-specific antibody interfered with the formation of the AMRE gel retardation complexes. The functional implications of the factors in AMOG gene regulation are discussed.
我们分析了新生大鼠大脑和小脑中原代培养星形胶质细胞中，调控神经胶质细胞黏附分子（AMOG）编码基因表达的顺式作用元件。在一系列5'端连续缺失突变体中，相对启动子活性与在B103（大鼠神经母细胞瘤细胞系）细胞中观察到的相似。先前鉴定的具有GAGGCGGGG序列的AMRE（AMOG调控元件）不仅在B103细胞中，而且在星形胶质细胞中均作为正调控元件发挥作用。基于使用星形胶质细胞和B103细胞的核提取物进行的以下观察，确定与该元件结合的因子为Sp1：（1）通过DNA酶I足迹法和甲基化干扰分析所分析的因子与AMRE的相互作用与Sp1相似；（2）因子与AMRE的结合与含有真实Sp1共有序列的寡核苷酸竞争；（3）Sp1特异性抗体干扰AMRE凝胶阻滞复合物的形成。讨论了这些因子在AMOG基因调控中的功能意义。
