STRUCTURAL AND FUNCTIONAL ANALYSIS OF A NEW CANDIDATE TUMOR-SUPPRESSOR GENE,DAN

新候选肿瘤抑制基因 DAN 的结构和功能分析

• 批准号: 07680754
• 负责人: OZAKI Toshinori
• 金额: $ 1.41万
• 依托单位: CHIBA CANCER CENTER RESEARCH INSTITUTE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680754/
• 关键词: DAN; DA41; Two-hybrid; Two-hybrid法; ゲノム遺伝子

(1) Characterization of the human DAN geneThe human genomic DNA,which contains the entire region of DAN gene, has been cloned from the standard phage- and the P1-based genomic libraries and its nucleotide sequence was determined. The human DAN gene is composed of four exons and there exist the possible CA-repeats within the first intron. Importantly, allelic loss of DAN gene locs was observed in three neuroblastoma tissues with N-myc gene amplification. At present, the molecular basis of the structural abnormalities of DAN gene in some of the neuroblastomas is not known.(2) ldentification of a new cellular protein which can associate with DANBy using a yeast two-hybrid screening, a new cellular protein (DA41) which can interact with DAN was identified. The DA41 cDNA encodes a new protein compised of 582 amino acids. The expression of DA41 gene was regulated in a cell cycle-dependent manner, raising a possibility that DA41 might possess a cell cycle-reglatory role. When DA41 gene was overexpressed in ras-transformed 3Y1 cells, phenotypic reversion was observed.Interestingly, the amount of WAF1 was increased and the activity of CDK2 was significantly reduced in these DA41-overexpressing cells. These results strongly suggest that the DAN-DA41 complex could contain a significant role in a cell cycle regulation.

(1)人DAN基因的表征从标准噬菌体和P1基因组文库中克隆了包含DAN基因整个区域的人基因组DNA，并测定了其核苷酸序列。人DAN基因由四个外显子组成，第一个内含子内可能存在CA重复序列。重要的是，在三个N-myc基因扩增的神经母细胞瘤组织中观察到DAN基因位点的等位基因丢失。目前，部分神经母细胞瘤中DAN基因结构异常的分子基础尚不清楚。(2)利用酵母双杂交技术筛选到一个新的与DAN相互作用的细胞蛋白DA 41。DA 41 cDNA编码一个由582个氨基酸组成的新蛋白。DA 41基因的表达具有细胞周期依赖性，提示DA 41可能具有细胞周期调节作用。在ras转化的3 Y1细胞中过表达DA 41基因后，细胞表型发生逆转，WAF 1表达量增加，CDK 2活性降低。这些结果有力地表明DAN-DA 41复合物可能在细胞周期调节中具有重要作用。

项目成果

Ozaki,T.,Hishiki,T.et al.: "Identification of a new cellular protein which can interact with DAN." DNA and Cell Biol.(in press). (1997)

Ozaki,T.,Hishiki,T.et al.：“鉴定出一种可以与 DAN 相互作用的新细胞蛋白。”

Ozaki, T. et al.: "Cloning of mouse DAN cDNA and its down-regulation in transformed cells." Jpn. J. Cancer Res.87. 58-61 (1996)

Ozaki, T. 等人：“小鼠 DAN cDNA 的克隆及其在转化细胞中的下调。”

Matsuda,Y.,Ozaki,T.,et al.: "Chromosome mapping of the mouse and rat homologues of the human DAN gene,D1S1733E." Mammalian Genome. 7. 709-710 (1996)

Matsuda,Y.,Ozaki,T.,et al.：“人类 DAN 基因 D1S1733E 的小鼠和大鼠同源物的染色体定位。”

Ozaki et al.: "Molecular cloning and characterization of a novel gene homologous to cdc37" DNA Cell Biol.14. 1017-1023 (1995)

Ozaki 等人：“与 cdc37 同源的新基因的分子克隆和表征”DNA Cell Biol.14。

Ozaki, T.Enomoto, H., Nakamura, Y., Kondo, K., Seki, N., Ohira, M., Nomura, N., Ohki, M., Nakagawara, A.and Sakiyama, S.: "The genomic analysis of human DNA gene." DNA and Cell Biol. (in press). (1997)

Ozaki, T.Enomoto, H.、Nakamura, Y.、Kondo, K.、Seki, N.、Ohira, M.、Nomura, N.、Ohki, M.、Nakakawara, A. 和 Sakiyama, S.：“