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Molecular interactions between Fanconi anemia pathway and familial breast cancer pathway mediated by BCCIP.

BCCIP 介导的 Fanconi 贫血途径与家族性乳腺癌途径之间的分子相互作用。

基本信息

批准号：
17590280
负责人：
ISHIAI Masamichi
金额：
$ 2.3万
依托单位：
Kawasaki Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590280/
关键词：
DNA repair Fanconi anemia familial breast cancer genetic disease DT40 yeast two-hybrid molecular biology genetics がん生化学

项目摘要

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 … More 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

Fanconi贫血（FA）通路与家族性乳腺癌易感基因（BRCA）通路之间的功能联系已有报道，但这些相互作用在体内的生理意义和确切的分子机制尚不清楚。通过使用鸡（ch）FancD 2的N-末端片段作为诱饵进行酵母双杂交（Y2 H）筛选，我们已经鉴定了BRCA 2相互作用蛋白chBCCIP的C-末端片段。为了证实这种相互作用，我们用表达His标记的全长chBCCIP和TAP标记的全长chFancD 2的质粒转染293 T细胞。TAP-FancD 2 pull-down和抗His蛋白质印迹法可以成功检测到这种相互作用。据报道，人类BCCIP与BRCA 2相互作用。为了证实FancD 2-BCCIP-BRCA 2相互作用，我们使用人cDNA进行Y2 H分析，但没有组合显示出显著的相互作用。然后，我们使用鸡cDNA进行哺乳动物双杂交（M2 H）试验。我们克隆了全长和原始片段 ...更多信息 ts用于载体中的Y2 H筛选，但我们不能检测到任何显著的相互作用测试组合。chBRCA 2片段的进一步表达不影响这些相互作用。这些结果表明，BCCIP和BRCA 2之间的相互作用不稳定和/或很弱。尽管我们分析了几百个克隆，但在鸡DT 40细胞中建立BCCIP基因条件靶向的尝试尚未成功。由于酵母BCIP基因是脊椎动物BCCIP的同源基因，因此BCCIP基因在DT 40细胞中的缺失可能导致细胞死亡。我们还尝试通过RNA干扰（RNAi）方法敲除人类细胞中的BCCIP基因。由于GFP-人BCCIP的表达水平在293 T细胞中通过共转染的RNAi显著降低，我们现在已经检查了降低内源性BCCIP基因表达的合适条件。在我们的相关研究中，我们报道了FANCC和BRCA 2基因在细胞生长和X射线敏感性方面具有上位性。这些结果表明FA和BRCA途径在DT 40细胞中存在遗传相互作用。需要进一步调查。少