Calzone F J, Höög C, Teplow D B, Cutting A E, Zeller R W, Britten R J, Davidson E H
Division of Biology, California Institute of Technology, Pasadena 91125.
Development. 1991 May;112(1):335-50. doi: 10.1242/dev.112.1.335.
The P3A2 regulatory protein interacts with specific sites in the control region of the CyIIIa actin gene. Previous studies showed that this interaction is required to confine expression of a CyIIIa.CAT fusion to the aboral ectoderm, the embryonic territory in which CyIIIa is normally utilized. P3A2 also binds specifically to similar target sites located in the regulatory region of the SM50 gene, which is expressed only in skeletogenic mesenchyme lineages. The P3A2 factor was purified by affinity chromatography from nuclear extracts of 24 h sea urchin embryos, and partial peptide sequences were used to isolate a cDNA clone encoding the complete protein. There are no significant similarities between P3A2 and any other protein in existing sequence data bases. P3A2 thus includes a novel type of DNA-binding domain. To examine the differential utilization of P3A2 in CyIIIa and SM50 genes, we measured the specific affinity of this protein for the various target sites in the regulatory DNAs of each gene, and identified the core target site sequences. The stability of P3A2 complexes formed with SM50 target sites is 50-100 times greater than that of the complexes formed with CyIIIa target sites, though the factor binds to very similar core sequence elements. P3A2 is one of at least twelve different proteins whose interaction with CyIIIa regulatory DNA is required for correct developmental expression. The results reported demonstrate that it might be possible to purify most of these regulatory proteins, or any other specific DNA-binding proteins of the sea urchin embryo, by using the simple procedures described for P3A2.
P3A2调节蛋白与CyIIIa肌动蛋白基因控制区域的特定位点相互作用。先前的研究表明，这种相互作用是将CyIIIa.CAT融合基因的表达限制在反口外胚层所必需的，反口外胚层是正常利用CyIIIa的胚胎区域。P3A2还特异性结合位于SM50基因调控区域的相似靶位点，该基因仅在成骨间充质谱系中表达。通过亲和层析从24小时海胆胚胎的核提取物中纯化出P3A2因子，并利用部分肽序列分离出编码完整蛋白的cDNA克隆。在现有的序列数据库中，P3A2与任何其他蛋白都没有显著的相似性。因此，P3A2包含一种新型的DNA结合结构域。为了研究P3A2在CyIIIa和SM50基因中的差异利用情况，我们测量了该蛋白对每个基因调控DNA中各种靶位点的特异性亲和力，并确定了核心靶位点序列。尽管该因子与非常相似的核心序列元件结合，但与SM50靶位点形成的P3A2复合物的稳定性比与CyIIIa靶位点形成的复合物高50 - 100倍。P3A2是至少十二种不同蛋白之一，其与CyIIIa调控DNA的相互作用是正确发育表达所必需的。所报道的结果表明，通过使用针对P3A2描述的简单程序，有可能纯化海胆胚胎中的大多数这些调控蛋白或任何其他特异性DNA结合蛋白。
