| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Apoptotic related genes, such as caspase-3, caspase-9, and Apaf-1, have a critical roles as cell death inducer. However, several evidences suggest that trivial activation of caspases, which do not induce cell death, needs to neural development and differentiation because knockout mice of caspase-3 and caspase-9 show disorganized neural development. In this project, we planned to identify caspases to be activated during neural development and screen its substrates to analyze the molecular roles of caspases.
Results
1.Identification of caspases expression during neural development.
The expression of caspase-7 was increased at early stage of primary neuronal culture by RT-PCR, whereas caspase-3 was increased during neural differentiation.
2.Generation of active caspase in E. coli.
Human caspase-3 and caspase-7 genes were cloned into pET expression vectors and active caspases were induced in E. coli., and purified.
3.Screening of substrates for caspase-7.
Small pool cDNA library contained about 100 genes from human brain per pool, which comes to 17,000 genes in total. Substrates were screened by in vitro cleavage assay in the presence of active caspase using 355-labeled proteins, which were generated by in vitro transcription and translation. Autoradiograph showed the cleaved product by caspase.
4.Identification of caspase-7 substrates from human brain cDNA.
Eighteen genes were identified as caspase-7 substrates from 96 pools cDNA library. Five of eighteen were unknown genes, ten genes included full-length, and two plasmids had no coding region. We are going to analyze the function of substrates for neural development.
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