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A study on the regulation of gene expression in sea urchin embryo.

海胆胚胎基因表达调控的研究。

基本信息

批准号：
02454022
负责人：
SHIMADA Hiraku
金额：
$ 4.35万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

In order to get an insight into the mechanism regulating gene expression during early development, we have started our research on the mechanism regulating the transcription of arylsulfatase (Ars) gene in the sea urchin embryo. Results of the present study are summarized as follows.(1) In addition to the minimal promoter containing a TATA sequence, the 5' flanking region of the Ars gene contains several transcription enhancer sequences in between -3kbp and -1.9kbp, and a transcription silencer sequence in between -1.5kbp and -1.3kbp.(2) Many proteins that bind to enhancer and silencer regions were detected by gel band mobility shify assay, and the nucleotide sequences within these regions that were protected by these proteins were determined by footprinting. Since patterns of appearance of these proteins in nuclei changed depending on developmental stages, it was assumed that these proteins are transacting factors for the Ars gene transcription.(3) The silencer region is a CT-rich region with nearly 90% of the bases in this region occupied by cytosine or thymine. It was revealed by Sl nuclease assay that a (CT)_<11> tract in this region can take a triplex DNA configuration that is expected to be a recognizing site for specific trans-acting factors. It was found by south-western blotting that several nuclear proteins bind to the silencer region, and some of them appear and disappear depending on developmental stages. We have already started the isolation of these DNA binding proteins.

为了深入了解海胆胚胎发育过程中基因表达的调控机制，我们对海胆胚胎中芳基硫酸酯酶（Ars）基因的转录调控机制进行了研究。本研究的结果总结如下。(1)Ars基因的5'侧翼区除了含有TATA序列的最小启动子外，还含有几个在-3kbp和-1.9kbp之间的转录增强子序列和一个在-1.5kbp和-1.3kbp之间的转录沉默子序列。(2)通过凝胶迁移率改变试验检测到许多与增强子和沉默子区域结合的蛋白质，并通过足迹法确定这些蛋白质保护的这些区域内的核苷酸序列。由于这些蛋白质在细胞核中的出现模式根据发育阶段而改变，因此假设这些蛋白质是Ars基因转录的反式作用因子。(3)沉默区是富含CT的区域，该区域中近90%的碱基被胞嘧啶或胸腺嘧啶占据。S1核酸酶分析表明，该区域的（CT）_<11>tract可形成三链DNA构型，可能是特异性反式作用因子的识别位点。通过西南印迹法发现，几种核蛋白与沉默区结合，其中一些蛋白的出现和消失取决于发育阶段。我们已经开始分离这些DNA结合蛋白。