Wegmann I S, Quack S, Spindler K D, Dorsch-Häsler K, Vögtli M, Lezzi M
Institut für Zellbiologie, Eidgenössische Technische Hochschule, Hönggerberg, Zürich, Switzerland.
Arch Insect Biochem Physiol. 1995;30(2-3):95-114. doi: 10.1002/arch.940300203.
Antisera were raised against different domains of a putative ecdysteroid receptor (cEcRH) of Chironomus tentans. All the antisera reacted with a 68,000 dalton protein exhibiting DNA binding properties. Additionally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [3H]ponasterone A, with high specificity. These properties indicate that the antisera recognize specifically an endogenous ecdysteroid receptor protein (cEcR) in C. tentans cells and thus are suitable for the following quantitative and qualitative immunological and immunohistochemical investigations. The cellular level of cEcR varies during development, and it is particularly low in oligopausing larvae. In polytene chromosomes of prepupal salivary glands, cEcR is located at approximately 50 transcriptionally active loci. These loci include both early ecdysteroid (Ec)-inducible puff sites, such as the locus containing the gene coding for the homolog of the E75 protein in Drosophila melanogaster, as well as late Ec-inducible puff-sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultraspiracle protein. However, loci of genes coding for salivary gland secretory proteins (e.g., Balbiani ring forming chromosome regions) do not specifically react with the antisera. Thus, the developmental regulation of these genes is not directly controlled by Ec. Polytene chromosomes of oligopausing larvae show hardly any loci that contain cEcR. The few detected correspond, with few exceptions, to the most potent cEcR binding sites found in prepupae.
制备了针对摇蚊假定蜕皮类固醇受体（cEcRH）不同结构域的抗血清。所有抗血清均与一种具有DNA结合特性的68,000道尔顿蛋白质发生反应。此外，我们能够证明抗血清免疫沉淀的蛋白质能与放射性标记的蜕皮类固醇（Ec），即[3H]蜕皮甾酮A特异性结合。这些特性表明抗血清能特异性识别摇蚊细胞中的内源性蜕皮类固醇受体蛋白（cEcR），因此适用于以下定量和定性的免疫及免疫组织化学研究。cEcR的细胞水平在发育过程中会发生变化，在寡停育幼虫中尤其低。在预蛹唾液腺的多线染色体中，cEcR位于大约50个转录活跃位点。这些位点包括早期蜕皮类固醇（Ec）诱导的胀泡位点，如含有果蝇E75蛋白同源基因编码的位点，以及晚期Ec诱导的胀泡位点。后一组包括一个指定果蝇超气门蛋白同源物的基因位点。然而，编码唾液腺分泌蛋白的基因位点（如形成巴尔比亚尼环的染色体区域）不会与抗血清发生特异性反应。因此，这些基因的发育调控不受Ec直接控制。寡停育幼虫的多线染色体几乎没有含有cEcR的位点。少数检测到的位点，除了少数例外，与预蛹中发现的最有效的cEcR结合位点相对应。
