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The Biological Functions of the SART-1 Family in Differentiation and Carcinogenicity of Adenocarcinomas

SART-1家族在腺癌分化和致癌性中的生物学功能

基本信息

批准号：
10670521
负责人：
SHICHIJO Shigeki
金额：
$ 2.18万
依托单位：
Kurume University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Molecules involved in regulation of cellular proliferation at the G2/M phase have not yet been fully identified. This study investigated the involvement of a recently identified SART-1ィイD2800ィエD2. Protein demonstrated an ability to bind to DNA, and accumulated in the nucleus when the cells were arrested at G2/M phase.(l) The localization of SART-1 protein was suggested to be in nucleus of eukaryote cells including monkey kidney EBV-transformed Cos 7 cell and human esophageal cancer TE9 cell when the fusion gene with GFP was transfected and observed by conforcal laser microscopy.(2) SART-1ィイD2800ィエD2 protein is suggested to be a DNA-binding protein by the affinity chromatography of cell lysates of the 293T cells transfected with the HAN/SART-1ィイD2800ィエD2 gene.(3) Transfection of SART-1 gene induced the G2/M-arrest and suppressed the cell growth of all the cell tested. Furthermore, DNA ladder formation was observed in the SART-1 transfected COS-7 and human cancer cells. The transfection of SART-1 up-regulated the nuclear expression of cyclin B and cdc2, well known components of maturation-promoting factor.(4) Several candidate genes which encode proteins that had interacted with SART-1 protein were isolated by two-hybrid system.(5) Three different SART-1 probe protein (N-terminal, middle and C-terminal regions) for far-western blot analysis of SART-1 binding protein candidate obtained by two hybrid system were prepared.(6) We found that murine SART-1 genomic DNA suggested to be consist of 20 exsons. The sequence of the gene will submit to GenBank.(7) SART-1 gene was suggested to be expressed in early development of mouse embryo (l4 days) by northern blot analysis.(8) Several family genes of SART-1 were suggested by northern blot analysis. One of the gene with 3.3kb consist of approximately l488 bp of 5'-region was identical to SART-1 and encoded a protein with 483 amino acid.

参与G2/M期细胞增殖调控的分子尚未完全确定。这项研究调查了最近发现的SART-1突变体D2800突变体D2的参与。蛋白质具有与DNA结合的能力，当细胞被阻滞在G2/M期时，蛋白质在细胞核中积累。(l)将SART-1与GFP融合基因转染到猴肾EBV转化的Cos 7细胞和人食管癌TE 9细胞中，激光共聚焦显微镜观察到SART-1蛋白定位于细胞核内。(2)通过对转染HAN/SART-1拟南芥D2800拟南芥D2基因的293 T细胞的细胞裂解物进行亲和层析，表明SART-1拟南芥D2800拟南芥D2蛋白是DNA结合蛋白。(3)转染SART-1基因的细胞均出现G2/M期阻滞，生长受到抑制。此外，在SART-1转染的COS-7和人癌细胞中观察到DNA梯状条带形成。SART-1的转染上调了细胞周期蛋白B和cdc 2的核表达，这是成熟促进因子的众所周知的组分。(4)利用双杂交技术分离了几个与SART-1蛋白互作的候选基因。(5)制备了三种不同的SART-1探针蛋白（N-末端、中间和C-末端区域），用于对通过双杂交系统获得的SART-1结合蛋白候选物进行远蛋白质印迹分析。(6)我们发现小鼠SART-1基因组DNA由20个外显子组成。该基因的序列将提交给GenBank。(7)北方印迹分析表明SART-1基因在小鼠胚胎早期（14天）表达。(8)通过北方杂交分析，推测了SART-1的几个家族基因。其中一个基因长3.3kb，5 ′端约1488 bp，与SART-1基因完全相同，编码483个氨基酸。