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.

为了深入了解早期发育过程中调节基因表达的机制，我们已经开始研究调节海胆胚胎中芳基磺化酶（ARS）基因转录的机制。（1）除了包含TATA序列的最小启动子外，ARS基因的5'侧翼区域还包含-3KBP和-1.9KBP之间的几个转录增强子序列，以及-1.5KBP和-1.3KBP之间的转录沉默序列（2）。通过凝胶带迁移率SHIFY分析检测区域，这些区域内受这些蛋白质保护的核苷酸序列是通过足迹确定的。由于这些核中这些蛋白质的外观模式取决于发育阶段，因此假定这些蛋白是ARS基因转录的交易因子。（3）消音器区域是一个富含CT的区域，该区域占该区域的近90％，在该区域占据了胞嘧啶或胸腺素。 SL核酸酶测定法表明，该区域中的A（CT）_ <11>可以采用三元DNA构型，该构型预计将是特定的跨性别因子的识别位点。西南印迹发现，几种核蛋白与消音区域结合，其中一些核蛋白会根据发育阶段出现并消失。我们已经开始分离这些DNA结合蛋白。