Hough-Evans B R, Franks R R, Zeller R W, Britten R J, Davidson E H
Division of Biology, California Institute of Technology, Pasadena 91125.
Development. 1990 Sep;110(1):41-50. doi: 10.1242/dev.110.1.41.
The CyIIIa.CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene.
将CyIIIa.CAT融合基因与过量的代表CyIIIa调控域亚区域的连接竞争序列一起注射到紫海胆卵中。在这个构建体中，氯霉素乙酰转移酶（CAT）报告基因置于谱系特异性CyIIIa细胞骨架肌动蛋白基因2300个核苷酸上游调控域的控制之下。通过原位杂交在源自注射卵的长腕幼虫期胚胎的连续切片中检测到CAT mRNA。当将缺乏竞争CyIIIa片段的载体DNA与CyIIIa.CAT共注射时，仅在反口外胚层细胞中观察到CAT mRNA，即CyIIIa基因本身正常表达的区域（我们之前也有报道）。当共注射七个带有DNA - 蛋白质相互作用位点的不同竞争亚片段中的五个时，得到了相同的结果。然而，共注射过量的CyIIIa调控域的两个广泛分离的非同源片段中的任何一个，都会在受体胚胎中产生CAT mRNA的显著异位表达。在这些胚胎的肠道、间充质细胞和口外胚层中观察到了CAT mRNA。我们得出结论，这些片段包含对CyIIIa基因空间表达起负调控作用的调控位点。
