M－CAT盒在鸡AchR　γ－亚基基因转录激活中的作用

The Role of M-CAT Boxes in Transcriptional Activation ofChick Acetylcholine Receptor γ-subunit Gene

原代培养鸡胚肌细胞瞬时转染结合氯霉素乙酰基转移酶水平测试表明：鸡ＡＣｈＲγ－亚基基因缺失近起始点一对Ｅ盒（ＣＡＮＮＴＧ）的－２０４／－５０片段与含Ｅ盒的－２０４／＋３６片段一样，均可独立激活ｔｋ启动子的转录活性，证明了该片段的增强子样作用，利用鸡胚肌肉核抽提物与－２０４／－５０片段进行胶阻滞分析，检出了明显的迁移位移条带的发生；迁移条带不仅可被未标记的－２０４／－５０片段消除，而且还可被含Ｍ－ＣＡＴ盒（ＣＡＴＴＣＣＴ）的２３ｂｐ寡核苷酸竞争阻断，说明胶阻滞分析中出现的迁移条带系由核内因子特异结合－２０４／－５０片段中的Ｍ－ＣＡＴ序列所致，上述结果证明，Ｍ－ＣＡＴ盒对鸡ＡＣｈＲγ－亚基基因－２０４／－５０片段的转录激活功能起作用。

It has previously been reported that CANNTG element (E box) and M-CAT motif present in chick acetylcholine receptor γ-subunit gene. Transient expression in primary culture cells and chloramphenicol acetyltransferase (CAT) assay indicated that-204/-50 fragment of this gene lacking a couple of E boxes near to transcription start site could activate heterologous TK promoter, and so did -204/ + 36 fragment. The mobility shift assays with -204/ -50 fragment and nuclear extracts from chick embryonic muscle showed shifted bands in 5% PAGE which could be specifically abolished by unlabeled-204/-50 fragment and a 23 bp oligonucleotide containing a M-CAT (CATTCCT) motif. These results demonstrated specific binding of M-CAT binding factor(MCBF) to-204/-50 fragment of γ-submit gene. It is likely that MCAT like-sequences may contribute to transcription activation of chick Ache γ-subunit gene.