GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

在一项工作中，我们继续研究核能 秀丽隐杆线虫的激素受体 之前克隆的。对于其中一种，我们用两种方法都展示了 抗体染色和与启动子连接的β-半乳糖苷 CHR3的增强子区域，在胚胎发育早期定位 到许多细胞的核，在胚胎发育后期和 线虫表皮细胞的发育。在这些细胞中， 蛋白质产物存在于细胞核中。因为转基因动物是 通过将RNA或DNA注射到卵巢内很容易形成 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，我们都能 为了证明完整的启动子/增强子活性，需要第一个1.6kb 起始点的上游，以及第一内含子的一部分。我们 也能够使用RNAi结构显示(在这些蠕虫中， 双链RNA(RNAi)是一种更有效的A 抑制Chr3功能的基因效应) 导致蠕虫显著死亡，而那些 存活下来的是未完全脱离的角质层。反向实验 其中CHR3过度表达的情况可以使用 热休克促进剂。这些虫子在下面都起了水泡 角质层。我们的结论是，CHR3是正常发展所必需的 尤其是对于换羽来说。我们不能确定这一影响是否 与蠕虫拥有的150个基因中的一个或多个有关 胶原蛋白，或者它是否与蛋白质分解功能有关。它是 感兴趣的是DHR3，一种与果蝇密切相关的同源物 那只昆虫的变态。我们目前正在努力 描述我们拥有的最原始的突变系之一 脊索动物，但已被证明可合成甲状腺素。卡洛萨医生 能够使用聚合酶链式反应技术分离出一种核激素 受体与脊椎动物的受体非常相似。奇怪的是，它并没有绑定 T4或T3。这可能是由于配体中的长序列造成的。 与任何其他LBD无关的结合域。这个基因， Cinr1仅在发育中的胚胎和幼虫中表达。 而成年人则不是。CnRN1在COS-7细胞中的表达 下调含有tre的报告基因。而且， 将鸡的tr、CnR-1基因导入同一细胞 以显性的负面方式抑制其表达。这 需要LBD。我们目前正在筛选其他CIONA图书馆 寻找更多的核激素受体，并已开始研究 扇贝体内的受体。

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.