Thézé N, Calzone F J, Thiebaud P, Hill R L, Britten R J, Davidson E H
Laboratoire de Génétique Moléculaire, CNRS, Universite de Rennes I, France.
Mol Reprod Dev. 1990 Feb;25(2):110-22. doi: 10.1002/mrd.1080250203.
Expression of the CyIIIa cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer experiments have defined a 2,300 nucleotide cis-regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA--protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high-specificity DNA--protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the CyIIIa regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., "CCAAT" and "octamer" elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.
CyIIIa细胞骨架肌动蛋白基因的表达是早期海胆胚胎反口外胚层中基因差异激活的一个标志。基因转移实验确定了一个2300个核苷酸的顺式调控结构域，该结构域对于该基因正确的空间和时间控制是必需的。这个结构域包括至少20个位点，在与胚胎核提取物反应时，体外会形成相对稳定的DNA-蛋白质复合物。我们报告了整个调控结构域的核苷酸序列，并绘制了发生高特异性DNA-蛋白质相互作用的位点图谱。这些位点最初是通过对大调控结构域给定亚片段的逐步限制性消化进行凝胶迁移分析定位的，然后通过寡核苷酸凝胶迁移竞争更精确地定位。特异性相互作用的位点中有8个在CyIIIa调控结构域内是独特的，其余的由5个不同的位点组成，这些位点出现不止一次。我们观察到一些在其他基因调控区域也发现的著名序列，例如“CCAAT”和“八聚体”元件。在其他研究中，根据假定的生物学功能对各种位点进行了分类，本研究允许评估构成每个功能类别的相互作用的数量和复杂性以及每个类别的位点的相对位置。
