Cascade of transcription factors in sea urchin early embryos

为了阐明早期发育过程中基因表达的机制，我们开始了一系列的研究，以找到一个级联的转录因子调节海胆胚胎Ars基因的表达。本课题获得的结果如下：我们以前报道过Ars基因转录起始位点附近的区域强烈地刺激该基因的启动子活性。今年，我们确定了顺式活性元件存在于这一地区的报告分析。发现两个顺式活性元件，一个在-72~-56 bp之间，与SpZI 2/OctI结合位点同源;另一个在+138~+142 bp之间，与Rel家族蛋白结合位点同源。两个顺式元件不仅刺激启动子活性，而且介导第一内含子增强子的刺激活性。除了这些元件之外，-194bp和-144bp之间的区域刺激Ars基因的时间调节表达。AACAAAG Strech在这一地区是一个结合位点的Sox（SRY型HMG盒）蛋白，一个已知的正转录因子，我们检测到的凝胶迁移试验的核蛋白从海胆原肠胚结合到这个基序序列特异性。这种蛋白质是一种母体蛋白质，存在于间充质囊胚期，但在原肠胚期后下降。海胆Sox蛋白在体外合成的SpSox mRNA结合序列特异性地这个主题。我们还检测到在近上游的Sox基序的存在的顺式元件，调节结合的Sox蛋白的Sox基序。

To elucidate a mechanism underlying gene expression during early development, we started a series of research to find a cascade for transcription factors regulating expression of the Ars gene of sea urchin embryos. Results obtained in the present project is as follows.We previously reported that the region around transcription start site of the Ars gene intensely stimulates the promoter activity of this gene. This year we determined cis-active elements present in this region by reporter assays. We found two cis-active elements, one between -72bp and -56bp is homologous to SpZI2/OctI -binding site and the other between +l38bp and +142bp homologous to the binding site for Rel family protein. Both cis-elements not only stimulate the promoter activity but mediate the stimulatory activity of the first intron enhancer. In addition to these elements, the region between -194bp and -144bp stimulate temporally regulated expression of the Ars gene. The AACAAAG strech in this region is a binding site for Sox (SRY-type HMG box) protein, a known positive transcription factor, and we detected by gel shift assay a nuclear protein from sea urchin gastrula that binds to this motif sequence-specifically. This protein is a maternal one that exists until mesenchyme blastulae but declines after gastrula stage. Sea urchin Sox protein synthesized in vitro by using SpSox mRNA bound sequence-specifically to this motif. We also detected in the near upstream of the Sox motif the existence of a cis-element that regulates binding of Sox protein to the Sox motif.