Genes Ancestral To The Thyroid/steroid Receptor Family

我们一直在研究锌和铜对核激素受体（NHRs）与DNA结合的影响以及对甲状腺激素调控转录的影响。结果表明，Zn和Cu在10-5 M左右浓度下对DNA和转录调控均有积极作用，但在更高浓度下对转录有负面影响。我们还观察到67Cu与水母RXR的DBD直接结合。我们的结果与NHRs的DBD可能在具有d轨道的二价离子的作用下获得第二种构象的可能性保持一致。这可能反映了这些离子在调节NHR功能中的生理作用，以及可能参与NHR功能机制的另一种构象，例如它们在受体循环过程中从DNA中释放出来。与二价金属调控发育的生理作用的可能性保持一致，铜的作用被铜的天然螯合剂组氨酸逆转。我们一直在研究一种酶的功能，活跃的组氨酸生产，肌肽酶在蠕虫。我们发现肌肽酶抑制导致发育停滞。我们目前正在研究蛋氨酸的功能，它在NHR的DNA结合域中是保守的，我们提出这种蛋氨酸可能是由于其侧链柔韧性而保守的，称为蛋氨酸翻转，这可能是NHR功能所必需的。我们还对秀丽隐杆线虫核激素受体CHR3的作用机制进行了研究，CHR3参与了脱毛的调控。我们已经证明CHR3是线虫发育中所有四种蜕皮的关键调节因子。为了寻找CHR3下游的基因，我们获得了一条表达dpy-7::gfp的细胞系，发现通过喂食引入CHR3 RNAi后，荧光明显降低。我们还发现SKIP的同源物是CHR3调控的一个辅助因子，最近证明该辅助因子参与秀丽隐杆线虫发育的多种调控途径。我们采用一种新的PCR定位线虫突变的方法，即片段- snp定位来确定molt-4突变的定位。通过这种方法，我们证实突变体molt-4位于染色体III上。我们继续对一种立方体水母进行研究。我们一直在实验室培养囊虫幼虫和含有眼睛的特殊结构——鼻鳞，我们正在寻找nhr。我们开始了一个小规模的cDNA测序项目。我们还在囊虫中发现了第二个NHR，目前正在尝试克隆它。该受体显然来源于nhr的RXR/RAR/TR亚家族。作为一种高度发育的水母，T. cystophora的眼睛既具有感光细胞又具有色素细胞，这对理解后生动物的进化具有重要意义。

We have been studying the effect of Zn and Cu on binding of Nuclear Hormone Receptors (NHRs)to DNA and effect on thyroid hormone regulated transcription. Our results show that both Zn and Cu have positive effect on both DNA and transcription regulation at concentrations around 10-5 M but affect negatively the transcription at higher concentrations. We have also observed direct binding of 67Cu to DBD of jellyfish RXR. Our results are keeping with the possibility that DBD of NHRs may be ble to quire a second conformation under the effect of divalent ions with d orbitals. This may reflect a physiological role of these ions in modulation of function of NHRs as well as the possibility of an alternative conformation which may be involved in mechanism of NHR function such as their release from DNA during the receptor recycling. Keeping with the possibility of physiological role of divalent metals in regulation of development, the effect of Cu was reversed by natural chelator of Cu, histidine. We have been studying the function of an enzyme active in histidine production, carnosinase in worms. We have found that carnosinase inhibition results in developmental arrest. We currently are studying the function of methionine, which is conserved in DNA binding domain of NHRs and we propose that this methionine may be conserved for its side chain flexibility termed methionine flipping, which is likely to be essential for NHR function. We have been also working on mechanism of action of CHR3, a nuclear hormone receptor in C. elegans, which is involved in regulation of molting. We have shown that CHR3 is a critical regulator of all four molts in C. elegans development. In search for genes downstream of CHR3, we have obtained a line expressing dpy-7::gfp and found that the fluorescence significantly decreased after introducing CHR3 RNAi by feeding. We have also identified that the homologue of SKIP is a cofactor in CHR3 regulation and recently proved that this cofactor is involved in multiple regulatory pathways in C. elegans development. We used a new method of mapping C. elegans mutations by PCR so called snip-SNP mapping to determine mapping of molt-4 mutation. With this method we confirmed that the mutant molt-4 is mapped to chromosome III. We continue our work on a cubmomedusan jellyfish T. cystphora. We have continual laboratory culture of T. cystophora larvae and specialized structures containing eyes - the rhopalia and we are searching for NHRs. We started a small scale of cDNA sequencing project. We have also identified second NHR in T. cystophora and we are currently trying to clone it. This receptor is apparently derived from RXR/RAR/TR subfamily of NHRs. T. cystophora, a highly developed jellyfish with eyes possessing single layer of cells which are photoreceptors as well as pigment cells seems to be important for understanding evolution of Metazoa.