Studies on the gene expression involved in the placetal development.

我们在芳香酶基因中发现了胎盘特异性增强子所必需的一个新的顺式元件。该元件和滋养层特异性元件(TSE)在芳香酶增强子区的共同存在足以指导由异源tk启动子驱动的滋养层特异性表达。在JEG-3细胞和人胎盘的核提取液中发现与该位点的结合活性，而在HeLa或Hep G2细胞的细胞提取液中未发现结合活性。这种结合活性不同于与芳香酶增强剂中的TSE样元件结合的蛋白质的凝胶迁移率。结合新的反式因子的核心DNA序列也不同于TSE结合蛋白的核心DNA序列。然而，在来自a-hCG启动子的24个碱基对的滋养细胞特异性元件(TSE)中发现了类似的序列，因此该DNA片段也以低亲和力与新因子结合。这个新的反式因子暂定名为TSEBP2.A2.65kb的cDNA克隆，编码436个氨基酸。该克隆是利用带有TSEBP2识别序列(GATCCATAAGACCCTCATTCCAGAG)的酵母单杂交系统从人胎盘cDNA文库中分离得到的。用杆状病毒系统将该克隆导入sf21细胞，获得了与JEG 3核提取液中的TSEBP2具有相同结合特异性的核蛋白。Northern-Blot分析表明，与该克隆对应的mRNA仅在胎盘中存在。该蛋白可能是滋养层细胞特异的转录因子，赋予胎盘特异性芳香酶基因的表达。然而，试探性反式激活结构域的一级结构与其他已知的转录因子没有相似之处。转录活性的证据正在得到证实。

We found a new cis-element neccessary for the placenta-specific enhancer in the aromatase gene. Co-presence of the element and the trophoblast specific element (TSE) in the aromatase enhancer region is sufficient to direct trophoblast specific expression driven by a heterologous tk promoter. The binding activity to this site was found in the nuclear extracts prepared from JEG-3 cells and the human placenta but not in the cell extract from HeLa or Hep G2 cells. This binding activity is distinct in electrophoreic mobility from the protein that binds to the TSE-like elements in aromatase enhancer. The core DNA sequence for the binding of the new trans-factor is also distinct from that of the TSE binding protein. The similar sequence is however found in the 24-base pair Trophoblast Specific Element (TSE) derived from the a-hCG promoter in reverse direction thus this DNA fragment also binds to the new factor with low affinity. This new trans-factor is tentatively called TSEBP2.A2.65 Kilobase cDNA clone that encodes a 436 amino acid was isolated from a human placental cDNA library using a yeast one-hybrid system with the TSEBP2 recognition sequence (GATCCATAAGACCCTCATTCCAGAG). The transfection of this clone with baculovirus system in sf21 cells resulted the production of a nuclear protein that showed the same binding specificity with the TSEBP2 in the nuclear extract prepared from Jeg 3. Northern-blot analysis showed that the mRNA coresponding to this clone was found exclusively in the placenta. This protein is likely to be a trophoblast specific transcription factor that confer placenta-specific expression of aromatase gene. The primary structure of the tentative transactivation domain however, has no similarity with other known transcription factors. The proof of transcriptional activity is on the way.