Studies on the complicating insulin-signaling of C.elegans based upon the ligand molecules

秀丽线虫的胰岛素信号调节滞育、寿命和脂肪代谢等多种现象。为了揭示复杂的胰岛素信号传导机制，我们对胰岛素样基因进行了干扰，并对其表型进行了观察。此外，构建了胰岛素样多肽和受体DAF-2的表达系统，以研究配体和受体的结合常数以及受体的磷酸化。利用UV/TMP方法对我们鉴定的类胰岛素基因CeIns-1和CeIns-2进行了干扰，突变体tm339缺失了CeIns-1基因的两个外显子，突变体tm790缺失了CeIns-2基因的一个外显子。这些突变体与野生动物杂交，以消除意想不到的突变。tm339的寿命略有延长，而tm790的寿命较短。在正常生长条件下，两个突变体均未进入滞育期。然而，I…在野生型动物培养滤液中制备的滞育诱导信息素存在的情况下，突变体tm339的滞育动物形成率明显高于野生型。相比之下，突变体tm790进入滞育期的速率较低。两个突变体表现出相反的表型，其中一个胰岛素样肽CeIns-3通过重组技术得到了表达。在大肠杆菌中以麦芽糖结合蛋白的形式表达，并用亲和层析的方法对其进行纯化。随后的酶处理将融合蛋白中的多肽释放出来，然后用反相HPLC对其进行纯化。为了构建DAF-2受体的表达系统，首先克隆了DAF-2的cDNA。RT-PCR扩增出多个daf-2的部分cDNA，并利用限制性内切酶切位点与这些cDNA结合，得到全长cDNAs。将信号肽的区域替换为人胰岛素受体的区域，然后将改变后的cDNA导入在哺乳动物细胞中发挥作用的表达载体中。较少

The insulin-signaling of the nematode Caenorhabditis elegans regulates various phenomena such as diapause, longevity, and fat metabolism. In order to elicit mechanism of the complicated insulin-signaling, the insulin-like genes were disrupted and the resulting phenotypes were observed. Moreover, expression systems of the insulin-like peptides and the receptor DAF-2 were constructed in order to investigate binding constant of the ligands and receptor and phosphorylation of the receptor.The insulin-like genes Ceinsulin-1 and -2, that we had identified, were disrupted by using the UV/TMP method The mutant named tm339 lost two exons in the Ceinsulin-1 gene, and the mutant designated tm790 lost one exon in the Ceinsulin-2 gene. The mutants were crossed with wild-type animals to remove unexpected mutation.The tm339 showed a little bit extended life span, while the tm790 exhibited a short life span. Both mutants did not enter into the diapause stage under a normal growth-condition. However, i … More n the presence of the diapuse-inducing pheromone prepared from culture filtrate of the wild-type animals, the mutant tm339 formed diapaused animals at remarkably higher rate. In contrast, the mutant tm790 entered into the diapause stage at quite lower rate. Both mutants showed opposite phenotypes.One of the insulin-like peptide Ceinsulin-3 was expressed by a recombinant technique. The peptide was expressed as a protein fused to a maltose-binding protein in E. coli, and the protein was purified by the method of affinity chromatography. The following protease treatment released the peptide from the fused protein, and then the peptide was purified by the reversed-phase HPLC.To construct an expression system of the receptor DAF-2, the daf-2 cDNA was cloned at first. RT-PCR yielded several partial cDNAs for daf-2, and the cDNAs were bound by utilizing restriction sites to produce the full-ledngth cDNA. The region for the signal peptide was substituted for that of human insulin receptor, and then the altered cDNA was introduced into an expression vector which functions in mammalian cells. Less