THE RETINOBLASTOMA SUSCEPTIBILITY GENE ENCODES A NUCLEAR PHOSPHOPROTEIN ASSOCIATED WITH DNA-BINDING ACTIVITY
THE RETINOBLASTOMA SUSCEPTIBILITY GENE ENCODES A NUCLEAR PHOSPHOPROTEIN ASSOCIATED WITH DNA-BINDING ACTIVITY
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DOI：
10.1038/329642a0
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发表时间：
1987-10-15
期刊：
影响因子：
64.8
通讯作者：
LEE, EYHP
中科院分区：
文献类型：
--
作者：
LEE, WH;SHEW, JY;LEE, EYHP
决定遗传性视网膜母细胞瘤易感性的人类基因（RB）最近已通过分子遗传学技术鉴定1，2，14。以前的结果表明，RB基因的完全失活是肿瘤形成所必需的。作为一种“抑癌基因”，RBthus的功能与大多数其他癌基因相反。RB互补DNA克隆的序列分析表明，一个长的开放阅读框编码一个假设的蛋白质，其特征提示DNA结合功能2。为了进一步证实和鉴定RB蛋白，我们制备了兔抗胰蛋白酶-RB融合蛋白的抗血清。纯化的抗RBIgG免疫沉淀蛋白质双联体，表观相对分子质量（Mr）为110，000 - 114，000。在所有表达正常RBmRNA的细胞系中均存在特异性蛋白，但在所检查的5个视网膜母细胞瘤细胞系中未检测到。RB蛋白能被~（32）P-磷酸代谢标记，表明它是一种磷蛋白。生化分级分离和免疫荧光研究表明，大部分的蛋白质位于细胞核内。此外，蛋白质可以保留和洗脱的DNA-纤维素柱，这表明它与DNA结合活性。综上所述，这些结果表明RB基因产物可能在调节细胞内的其他基因中起作用。
The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques1,2,14. Previous results indicate that complete inactiva-tion of theRBgene is required for tumour formation. As a 'cancer suppressor' gene,RBthus functions in a manner opposite to that of most other oncogenes. Sequence analysis ofRBcomplementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function2. To further substantiate and identify theRBprotein, we have prepared rabbit antisera against a trypE-RBfusion protein. The purified anti-RBIgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000–114,000. The specific protein(s) are present in all cell lines expressing normalRBmRNA, but are not detected in five retinoblastoma cell lines examined. TheRBprotein can be metabolically labelled with32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that theRBgene product may function in regulating other genes within the cell.