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

秀丽隐杆线虫的胰岛素信号调节各种现象，如滞育、长寿和脂肪代谢。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破坏，并观察了由此产生的表型。此外，我们构建了胰岛素样肽和受体DAF-2的表达系统，以研究配体与受体的结合常数和受体的磷酸化情况。用紫外/TMP方法对已鉴定的胰岛素样基因Ceinsulin-1和-2进行了破坏，突变体tm339在Ceinsulin-1基因上丢失了两个外显子，突变体tm790在Ceinsulin-2基因上丢失了一个外显子。这些突变体与野生型动物杂交，以去除意想不到的突变。tm339的寿命略有延长，而tm790的寿命较短。两个突变体在正常生长条件下均未进入滞育期。然而，在野生型动物培养滤液制备的诱导滞育信息素存在的情况下，突变体tm339的滞育率显著提高。而突变体tm790进入滞育期的速率较低。两个突变体表现出相反的表型。用重组技术表达了其中一种胰岛素样肽Ceinsulin-3。该肽在大肠杆菌中表达为与麦芽糖结合蛋白融合的蛋白，并用亲和层析法纯化该蛋白。接下来的蛋白酶处理从融合蛋白中释放肽，然后用反相高效液相色谱纯化肽。为了构建受体DAF-2的表达体系，首先克隆DAF-2 cDNA。RT-PCR得到了daf-2的几个部分cDNA，这些cDNA通过限制性内切位点结合产生全长cDNA。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导入到哺乳动物细胞的表达载体中。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