STUDIES OF TRANSCRIPTION FACTOR WHICH REGULATE TISSUE AND CELL SPECIFIC EXPRESSION OF hCG mRNA

1.我们用hCG基因转染小鼠Y1细胞和CHO细胞。这些细胞一旦转染hCG基因就开始表达hCG。我们从这些转染的细胞以及从人滋养层细胞系JAr和JEG3，以及人非滋养层细胞系HeLa中提取核蛋白。我们用这些细胞的核提取物进行足迹分析，以确定hCG转录因子的结合位点，我们发现这些细胞的足迹没有差异。提示hCG的组织特异性转录受一个不直接与hCG基因结合的因子调控，而是受一个影响与hCG基因结合的因子活性的因子调控.我们制备了在hCG转录调控元件中具有突变的克隆，并鉴定了转录因子结合所必需的序列。我们还进行了寡核苷酸竞争实验，鉴定了转录因子的结合位点.新的转录因子被认为是激活hCG基因的关键。

1. We transfected mouse Y1 cells and CHO cells with hCG genes. These cells started to express hCG once transfected with hCG gene. We extracted nuclear protein from these transfected cells as well as from human trophoblast cell lines JAr and JEG3, and also human non trophoblast cell line HeLa. We used nuclear extracts from these cells for footprint assay to identify the binding site for transcription factor for hCG.We found no difference in the footprint among these cells. Thus it was suggested that tissue specific transcription of hCG is regulated by a factor not directly bound to hCG gene, but rather a factor which affect the activity of the factors bond to the hCG gene.2. We made clones which have mutation in hCG transcription regulatory element and identified sequences necessary for binding of transcription factors. We also performed competition assay with oligonucleotides and identified binding sites of transcription factors.3. New transcription factors were suggested for the activation of hCG genes.