| Project/Area Number |
16043223
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Kyoto University |
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Principal Investigator |
MURAMATSU Masamichi Kyoto university, Graduate school of medicine, COE associate professor, 医学研究科, COE助教授 (20359813)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥26,400,000 (Direct Cost: ¥26,400,000)
Fiscal Year 2006: ¥8,800,000 (Direct Cost: ¥8,800,000)
Fiscal Year 2005: ¥8,800,000 (Direct Cost: ¥8,800,000)
Fiscal Year 2004: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Keywords | Antibody / hypermutation / class switch / activation induced deaminase / immunology / 抗体 / siRNA / hypermutation / 体細胞変異 / B細胞 / RNA editing / 組み換え / 免疫グロブリン |
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| Research Abstract |
We previously revealed that activation induced cytidine deaminase (AID) is essential and control two genetic alteration systems namely immunoglobulin class switch recombination and somatic hypermutation. Still it is elusive how AID undergoes and differentially controls such two events. In order to understand how class switch recombination and somatic hypermutation are regulated by AID, we are trying to identify cofactors for AID. We took two approaches to raise possible candidates for AID binding protein. One is two hybrid screening using AID and AID mutants as baits. After cDNA screening of libraries, we isolated 30 candidates that are expressed in 8 cells and physically interact with AID protein at the level of two-hybrid assay. The other approach was co-immunoprecipitation using mammalian cell lines that express AID tagged with flag peptide. AID complex was enriched by the immunoprecipitation and resolved by SDS-PAGE, and then AID or AID mutant-specific precipitants were decided by mass analysis. Previous studies provided two kinds of AID mutants (class switch defective-and somatic hypermutation defective mutants). Class switch defective AID mutants do not have 16 amino acids at C-terminus. On the other hand, somatic hypermutation defective AID mutants do have missense mutation an N-terminus. The two mutants were applied to take AID binding protein. Based on expression in B cells, mode of binding with AID/mutants and cDNA information, we took eight candidates as reasonable and possible AID cofactors. Now to see functional significance for the candidates, we limit expression of candidates by transduction of siRNA. When we knock down expression of one particular candidate, we observed 90% reduction of class switch inducing activity. Further study of this candidate should be done to know why class switch inhibition is observed by reducina expression level of the candidate.
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