GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

在我们之前从秀丽隐杆线虫中克隆的受体CHR3的继续工作中，我们使用RNAi浸泡动物的技术，我们发现这对幼虫阶段L2和L3没有影响。先前的研究表明，将RNAi注射到成虫体内会产生不完全的蜕皮，但表皮仍然附着在动物身上。可能只有某些蜕皮才需要CHR3。为了了解是否其他基因也可能参与换羽，我们对3000只蠕虫进行了诱变，发现一种突变导致了换羽缺陷，这种缺陷与缺乏CHR3导致角质层不完全脱落的缺陷相似。这个基因定位在3号染色体上，因此与1号染色体上的CHR3不同。我们正在更精确地定位这个基因，然后从现有的序列数据将能够识别它并研究它的作用机制。我们之前研究了CHR3的表达和过表达。我们现在有更多的数据表明，通过注射大量CHR3 DNA过度表达会导致额外的缝细胞、额外的胚胎，有时甚至会导致胚胎畸形。这些影响在包括启动子在内的整个基因中都可以看到，在LBD被删除时也是如此。未来的计划包括比较c briggsae和c vulgaris线虫中CHR3的启动子序列，这两种线虫有一定的亲缘关系，以确定哪些序列是保守的，因此可能是控制基因活性的主要位点。正在进行的其他工作将试图确定果蝇中似乎由DHR3控制的Ftz Fl是否由hchr3调节。在哺乳动物中，CHR3也与ROR/RZR同源，它们影响一些血凝域蛋白的转录。在秀丽隐杆线虫中，egl-5负责尾部发育我们将看到CHR是否调节其转录。(Drs。Kostrouchova和Krause)。在另一项调查中，我们研究了水母，tripedalia cystophora。去年，我们从这个物种中克隆了一个核激素受体基因，jRXR，它与脊椎动物的RXR基因非常相似。我们还发现它与由Joram Piatigorsky克隆的三个水母晶状体结晶蛋白基因的某些启动子序列结合。现在我们已经证明，在兔晶状体细胞培养系统中，jRXR使连接到结晶蛋白启动子序列的荧光素酶的表达增加了4到10倍。类维生素a对这种增加的影响是不可重复的。在非晶状体组织，如NIH 3T3细胞或小鼠胚胎癌细胞中未见效果。Western blotting法在三足动物眼中检测到JRXR。令人惊讶的是，EM技术发现，jRXR的主要染色是在色素颗粒中。针对TR和RAR亚家族不同结构域的抗体的进一步研究得到了积极的结果，因此我们目前正试图从三足植物中克隆核激素受体家族的其他成员。为了在三足动物中寻找内源性类维生素a，我们在dr。Wiggert和jRXR能够初步显示所有反式维甲酸的存在（jRXR仅结合9个顺式维甲酸）。计划进一步的实验来解决这个问题。(Drs。Kostrouch和Piatigorsky)。在另一项相关研究中，我们检测了软体动物麦哲伦Plactopecten magellenicus和辐照农胶中的核激素受体。利用RT-PCR技术，我们获得了三个长度约为150 bp的不同片段的最保守的DBD区域。SC5与孤儿受体TR2-11具有76%的同源性。SC7与CF1/USP的同源性为92%，SC8与sevenup的同源性为92%，SC11与E75的同源性为90%。这些都是核激素受体。USP是RXR的果蝇同源物，与EC受体结合蜕皮激素。E75是由蜕皮激素开启的，而7up则参与感光细胞的发育。看来，眼睛的发育受到核激素受体的影响，就像变态一样——至少在果蝇身上是这样。我们现在正试图获得这些基因的完整序列，并将研究它们的功能。(Drs。Carosa Piatigorsky)。-秀丽隐杆线虫，表皮细胞，刺胞体，荚膜，核激素受体

In a continuation of work on the receptor CHR3 which we previously cloned from c elegans, using a technique of soaking the animals with RNAi, we found that this had no effect on the larval stages, L2 and L3. Previous studies showed that RNAi injected into adult worms produced incomplete molting with the cuticle still attached to the animal. It may be that CHR3 may only be required for certain molts. To see if other genes may also be involved in molting we mutagenized 3,000 worms and found one mutation that caused a molting defect similar to that caused by a lack of CHR3 with incompletely detached cuticle. This gene has been localized to Chromosome 3 and hence is different than CHR3 which is on chromosome 1. We are in the process of localizing this gene more precisely and then from the sequence data available will be able identify it and study its mechanism of action. We had previously investigated expression and overexpression of CHR3. We now have additional data which shows that overexpression by the injection of large amounts of CHR3 DNA causes extra seam cells, additional alae and sometimes deformed embryos. These effects were seen with entire gene including its promoter and also when the LBD had been deleted. Future plans include a comparison of the promoter sequences of CHR3 in c briggsae and c vulgaris, somewhat distantly related nematodes to determine which sequences are conserved and hence likely to be major sites for control of gene activity. Other work which is under way will attempt to determine whether Ftz Fl which in the drosophila appears to be controlled by DHR3, is modulated byh CHR3. CHR3 is also homologous to ROR/RZR in mammals which affect transcription of some hemeodomain proteins. In c elegans egl-5 is responsible for tail development and we will see if CHR regulates its transcription. (Drs. Kostrouchova and Krause).In another investigation, we have studied the jelly fish, tripedalia cystophora. Last year we cloned a nuclear hormone receptor gene, jRXR from this species which is remarkably homologous to the vertebrate RXR gene. We also showed that it binds to certain promoter sequences in three jellyfish lens crystallin genes cloned by Joram Piatigorsky. Now we have shown that in a rabbit lens cell culture system, jRXR increased 4 to 10 fold the expression of luciferase which had been ligated to the crystallin promoter sequences. The effect of retinoids on this increase was not reproducible. No effect could be demonstrated in non lens tissues, such as NIH 3T3 cells or mouse embryonal carcinoma cells. JRXR could be detected by Western blotting in the rhopalia (eyes) of tripedalia. Surprising was the finding that with EM techniques, the main stain for jRXR was in pigment granules. Further studies with antibodies to different domains of the TR and RAR subfamilies gave positive results so we are currently attempting to clone additional members of the Nuclear hormone family of receptors from tripedalia. To search for endogenous retinoids in tripedalia, we examined larvae with help fromDr. Wiggert and were able tentatively to show the presence of all trans retinoic acid (jRXR binds only 9 cis retinoic acid). Further experiments are planned to get this straightened out. (Drs. Kostrouch and Piatigorsky).In another related study, we have examined nuclear hormone receptors in the mollusc Plactopecten magellenicus and agropectin irradians. Using RT-PCR we obtained the most consersed DBD regions of three different fragments about 150 bp in length. SC5 showed 76% identity with the orphan receptor TR2-11. SC7 showed 92% identity with CF1/USP, SC8 had 92% identity with seven-up and SC11 had 90% identity with E75. All of these are nuclear hormone receptors. USP is the drosophila homologue of RXR and with the EC receptor binds ecdysone. E75 is turned on by ecdysone and seven up is involved in photoreceptor cell development. It would appear that eye development is influenced by nuclear hormone receptors as is metamorphosis-- at least in drosophila. We are now attempting to get complete sequences of these genes and will study their function. (Drs. Carosa, Piatigorsky). - caenorhabditis elegans,epidermal cells, cnidaria, ciona intestinalis, nuclear hormone receptors