GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

在一项工作中，我们继续核 从秀丽隐杆线虫中提取的激素受体 以前克隆的其中之一，1993年，我们展示了这两个与 抗体染色和β半乳糖苷连接到启动子 CHR 3的增强子区域，在早期胚胎发生中，它定位于 许多细胞的细胞核，并在胚胎发育后期和 在蠕虫的表皮细胞中发育。在这些细胞中， 蛋白质产物存在于细胞核中。由于转基因动物是 通过将RNA或DNA注射到卵巢中， 蠕虫的合胞体，并且表达可以用 无论是绿色荧光蛋白还是β半乳糖苷， 为了显示完整的启动子/增强子活性需要前1.6kb的启动子/增强子序列， 起始位点的上游，以及第一内含子的一部分。我们 也能够显示使用RNAi构建体（在这些蠕虫中， 双链RNA（RNAi）是一种更有效的抑制剂， 基因的作用比反义RNA的作用更强 导致了蠕虫的显著死亡率，而那些 幸存者的表皮没有完全脱落反向实验 其中CHR 3过表达可以使用 热激促进剂这些蠕虫在皮肤下显示出水泡， 角质层我们的结论是，CHR 3是必要的适当发展 特别是用于蜕皮。我们无法确定是否影响 与蠕虫的150个基因中的一个或多个有关， 胶原蛋白，或者是否与蛋白水解功能有关。具有 令人感兴趣是，DHR 3是一种与果蝇密切相关的同源物， 昆虫的变态我们目前正试图 描述一个突变系，我们有一个最原始的 脊索动物，但已被证明能合成甲状腺素。卡罗萨博士 能够利用PCR技术分离出一种核激素 与脊椎动物TR非常相似的受体。奇怪的是， T4或T3。这可能是由于配体中的长序列 与任何其他LBD无关的结合结构域。这个基因 ciNR 1仅在发育中的胚胎和幼虫中表达 而不是在成人中。CoS 7细胞中CiNR 1的表达导致 下调含有TRE的报告基因。尿急， 当同样的细胞转染鸡TR，CiNR 1 以显性负性方式抑制其表达。这 需要LBD。我们目前正在筛选其他ciona图书馆 并开始研究 扇贝中的受体

In one line of work we continued with nuclear hormone receptors from Caenorhabditis elegans which we had previously cloned. With one of them, CHR 3 we showed both with antibody staining and with beta galactoside ligated to the promoter enhancer region of CHR3, that in early embryogenesis it localized to the nucleus of many cells, and in late embryogenesis and during development in epidermal cells of the worm. In these cells the protein product is found in the nucleus. Since transgenic animals are easily developed by injection of RNA or DNA into the ovarial syncytium of the worm, and expression can be monitored with either green fluorescent protein or beta galactosides, we were able to show that full promoter/enhancer activity required the first 1.6kb upstream of the start site, as well as a portion of the first intron. We were also able to show using an RNAi construct (in these worms, double stranded RNA (RNAi) is a more effective inhibitor of a gene's effect than antisense RNA) that inhibiting CHR 3 function resulted in a significant mortality in the worms, and those that survived had incompletely detached cuticle. The reverse experiment in which CHR3 was over expressed could be performed using a heat shock promoter. These worms showed blisters under the cuticle. We conclude that CHR3 is required for proper development and in particular for molting. We cannot be sure whether the effect is related to one or more of the 150 genes the worm has for collagens, or whether it is related to a proteolytic function. It is of interest that DHR3, a close drosophila homologue is related to metamorphosis in that insect. We are currently trying to characterize a mutant line we have one of the most primitive of chordates but has been shown to synthesize thyroxine. Dr. Carosa was able using the PCR technique to isolate a nuclear hormone receptor quite similar to vertebrate TRs. Curiously, it did not bind T4 or T3. This was probably due to a long sequence in the ligand binding domain that is unrelated to any other LBD. This gene, ciNR1 was only expressed in the developing embryo and the larva and not in the adult. Expression of CiNR 1 in COS 7 cells caused down regulation of a reporter gene containing a TRE. urthermore, when the same cells were transfected with a chicken TR, CiNR 1 inhibited its expression in a dominant negative fashion. This required the LBD. We are currently screening other ciona libraries for further nuclear hormone receptors and have begun work on receptors in scallops.