| Project/Area Number |
23710251
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Living organism molecular science
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
NOMURA Wataru 東京医科歯科大学, 生体材料工学研究所, 准教授 (80463909)
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2011: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
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| Keywords | 生体分子の化学修飾 / 蛋白質 / 遺伝子 / ゲノム / メチル化 / エピジェネティクス / タンパク質 / DNA / ジンクフィンガー |
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| Research Abstract |
Covalent modification of DNA, such as cytosine methylation, can induce heritable gene silencing. If epigenetic modifications can be specifically targeted, new approaches to transcriptional therapy should result. To address this challenge we have constructed methyltransferases that would act only at a desired site by adapting the sequence-enabled assembly strategy. This is the first successful application of the sequence-enable enzyme reassembly approach in vivo. In this study, to determine the functions of split protein domains in DNA binding and methylation, the split domains were expressed and purified separately. Utilizing these domains, DNA binding analyses were performed. The results indicate cooperative binding of the domains to the specific DNA targets. This interaction between the domains shows a direct evidence of assembly on the target sequence of split domains. The complementary protein assays have been shown their usefulness in dissection of protein interaction in mammalian cells. However, a few of direct approach to evaluate kinetics of interaction of split domains have been performed. Moreover, to expand the targetable DNA sequences on genomic DNA, several zinc finger domains were constructed. These domains showed DNA binding on endogenous targets. To perform efficient DNA methylation in mammalian cells, the expression of methyltransferase would be a key factor. Thus, the codon usage of methyltransferase was optimized. The expression of methyltransferase was greatly increased. The present results would expand the knowledge in the design of split protein domains.
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