| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We made an expression construct that expresses VP16-fused P53 protein followed by exploration of the interaction partners (Gal4-fusion proteins) using the mammalian two-hybrid methods (M2H). We detected two significant partner candidates judging from luciferase reporter activity ; one was mdm2, a known P53 binding partner, and the other was a 17 kDa protein without known functions. We tried co-immuno-precipitation using the tagged proteins to confirm the M2H result, although we did not confirm the latter association. Because we developed a novel method for rapid in vitro pull-down assay, we applied the method for the confirmation. However, we could not confirm the latter interaction.
Next we systematically explored protein-protein interactions among mouse transcription factors. For this purpose, we used approximately 1,700 mouse cDNAs for transcription factors/transcription regulation factors. The self-activation experiment showed that 14% of transcription factors have trans-activation activity as Gal4-fusion proteins, which was significantly higher than 2-3% of trans-activation activity for other proteins. We found approximately 4,000 protein-protein interactions using the M2H. The result showed that one interaction was identified per 720 trials, which was also significantly higher value than normal case (one per thousands). Generally, there are many false-positive interactions in the two-hybrid data. Therefore, we validated our result using in vitro pull-down assay described above ; the validation was successful with very high rate (90%) for the publicly known interactions, and was also partially successful (60%) for the unknown interactions. Thus we could extract the true-positive protein-protein interactions as much as possible.
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