| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Functional linkage between Fanconi anemia (FA) pathway and familial breast cancer susceptibility gene (BRCA) pathway has been reported, but physiological significance and exact molecular mechanism of these interactions in vivo have not been identified. By performing yeast two-hybrid (Y2H) screening using N-terminal fragment of chicken (ch) FancD2 as a bait, we have identified C-terminal fragment of chBCCIP, a BRCA2-interacting protein. To confirm this interaction, we transfected 293T cells with plasmids, which express His-tagged full-length chBCCIP and TAP-tagged full-length chFancD2. TAP-FancD2 pull-down and anti-His Western blotting could successfully detect this interaction. Human BCCIP was reported to interact with BRCA2. To confirm FancD2-BCCIP-BRCA2 interaction, we performed Y2H analysis using human cDNAs, but none of the combinations showed significant interaction. Then, we performed mammalian two-hybrid (M2H) assay using chicken cDNAs. We cloned full-length and original fragmen
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ts used in Y2H screening in the vectors, but we could not detect any significant interactions tested combinations. Further expression of chBRCA2 fragments did not affect to these interactions. These results suggested that interactions between BCCIP and BRCA2 were not stable and/or very week. Attempt for establishing the conditional targeting of BCCIP gene in chicken DT40 cells has not been successful, even though we analyzed several hundred clones. Since yeast BCIP gene, a homolog of vertebrate BCCIP, is an essential gene, BCCIP deletion in DT40 cell could lead to lethality. We also tried to knock down the BCCIP gene in human cells by RNA interference (RNAi) method. Since expression levels of GFP-human BCCIP was markedly reduced by co-transfected RNAi in 293T cells, we have now examined the suitable conditions for reduction of endogenous BCCIP gene expression. In our related study, we reported that FANCC and BRCA2 gene epistatic regarding cell growth and sensitivity to X-ray using double knock out DT40 cells. These results indicated that FA and BRCA pathways interact genetically in DT40 cells. Further investigations would be warranted. Less
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