| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Research Abstract |
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
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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
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