Bevilacqua A, Kinnunen L H, Bevilacqua S, Mangia F
Department of Psychology, La Sapienza University of Rome, Italy.
Dev Biol. 1995 Aug;170(2):467-78. doi: 10.1006/dbio.1995.1230.
In early mouse embryos, the major inducible heat shock gene, hsp68, is spontaneously and transiently activated at the two-cell stage and becomes heat-inducible around blastocyst stage. We have probed mouse embryo's ability to activate the promoter of this gene during preimplantation development by expression analysis of DNA constructs containing a reporter lacZ gene driven by hsp68 (hsp70A1) 5'-regulatory sequences of various length: (i) a full-length promoter (construct phsplacZ); (ii) a heat shock element (HSE)-deleted promoter (p delta 1hsplacZ); and (iii) a minimal, proximal promoter (p delta 2hsplac Z). When analyzed in transfected L-cells, phsplacZ was heat-inducible, while neither p delta 1hsplacZ nor p delta 2hsplacZ was. Developmental activity of the full-length construct was first analyzed after genome integration in transgenic embryos and found to follow endogenous hsp68 expression in terms of spontaneous activation at the 2-cell stage, down-regulation at the 4-cell stage, and acquisition of heat inducibility at the 16/32-cell stage. In transient expression experiments, injected phsplacZ, p delta 1hsplacZ, and p delta 2hsplacZ were expressed at similar levels by 2-cell embryos, independently of construct topology and injection stage. At the 4-cell stage, however, phsplacZ and p delta 1hsplacZ were expressed at similar levels, while p delta 2hsplacZ was inactive. Only phsplacZ became heat-inducible in late morulas. We conclude that in early mouse embryos, developmental activity of episomic hsp68 promoter depends on proximal sequences at the 2-cell stage and on putative enhancer sequences at the 4-cell stage, while HSEs appear dispensable during early cleavage.
在早期小鼠胚胎中，主要的可诱导热休克基因hsp68在二细胞阶段自发且短暂地被激活，并在囊胚阶段左右变得可被热诱导。我们通过对含有由不同长度的hsp68（hsp70A1）5'调控序列驱动的报告基因lacZ的DNA构建体进行表达分析，来探究小鼠胚胎在植入前发育过程中激活该基因启动子的能力：（i）全长启动子（构建体phsplacZ）；（ii）缺失热休克元件（HSE）的启动子（p delta 1hsplacZ）；以及（iii）最小的近端启动子（p delta 2hsplacZ）。在转染的L细胞中进行分析时，phsplacZ可被热诱导，而p delta 1hsplacZ和p delta 2hsplacZ均不能。在转基因胚胎中进行基因组整合后，首先对全长构建体的发育活性进行了分析，发现其在二细胞阶段的自发激活、四细胞阶段的下调以及16/32细胞阶段获得热诱导性方面均与内源性hsp68表达一致。在瞬时表达实验中，注射的phsplacZ、p delta 1hsplacZ和p delta 2hsplacZ在二细胞胚胎中以相似水平表达，与构建体拓扑结构和注射阶段无关。然而，在四细胞阶段，phsplacZ和p delta 1hsplacZ以相似水平表达，而p delta 2hsplacZ无活性。只有phsplacZ在晚期桑椹胚中变得可被热诱导。我们得出结论，在早期小鼠胚胎中，游离型hsp68启动子的发育活性在二细胞阶段取决于近端序列，在四细胞阶段取决于假定的增强子序列，而HSE在早期卵裂过程中似乎是可有可无的。
