MUTATION IS REQUIRED TO ACTIVATE THE P53 GENE FOR COOPERATION WITH THE RAS ONCOGENE AND TRANSFORMATION
MUTATION IS REQUIRED TO ACTIVATE THE P53 GENE FOR COOPERATION WITH THE RAS ONCOGENE AND TRANSFORMATION
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DOI：
10.1128/jvi.63.2.739-746.1989
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发表时间：
1989-02-01
影响因子：
5.4
通讯作者：
LEVINE, AJ
中科院分区：
文献类型：
--
作者：
HINDS, P;FINLAY, C;LEVINE, AJ
先前的实验已经提出了问题，p53癌基因产物的哪个氨基酸序列应该被认为是野生型，以及正常蛋白质是否能够与ras癌基因合作转化培养中的细胞。为了解决这些问题，一系列的p53 cDNA-基因组杂交克隆的能力进行了比较，合作的ras癌基因的转化试验。从这些实验中可以清楚地看出，在基因组克隆或cDNA克隆中，第135位的氨基酸丙氨酸不能产生与ras癌基因和转化细胞协同作用的p53蛋白。在该测定中，在基因组或cDNA克隆中的该位置处用缬氨酸替换丙氨酸以激活转化。使用p53基因中的限制性酶多态性，结果表明正常小鼠DNA编码p53蛋白第135位的丙氨酸。因此，需要突变来激活p53蛋白以与ras癌基因合作。与激活的ras基因共转染后，基因组p53 DNA克隆总是产生更多的转化细胞灶（1.7倍）比类似的cDNA克隆，这些病灶更容易克隆（3.6倍）到永久性细胞系。采用基因组p53克隆的一系列缺失突变体来显示p53基因中内含子4的存在足以提供来自培养皿的转化灶的大大增强的克隆能力。p53基因结构中内含子的存在也导致p53-plus-ras-转化细胞系中p53蛋白水平升高。因此，需要p53蛋白质的质的变化来激活p53以与癌基因ras转化。转化频率的定量改善与通过在转化质粒中具有p53内含子之一而提供的改变的p53蛋白的更高表达水平相关。
Previous experiments have brought into question which amino acid sequence of the p53 oncogene product should be considered wild type and whether the normal protein is capable of cooperating with the ras oncogene to transform cells in culture. To address these questions, a series of p53 cDNA-genomic hybrid clones have been compared for the ability to cooperate with the ras oncogene in transformation assays. From these experiments, it has become clear that the amino acid alanine at position 135, in either the genomic clone or the cDNA clone, failed to produce a p53 protein that cooperated with the ras oncogene and transformed cells. Replacing alanine with valine at this position in either the genomic or the cDNA clone activated for transformation in this assay. Using restriction enzyme polymorphisms in the p53 gene, it was shown that normal mouse DNA encodes alanine at position 135 in the p53 protein. Thus, mutation is required to activate the p53 protein for cooperation with the ras oncogene. After cotransfection with the activated ras gene the genomic p53 DNA clone always produced more transformed cell foci (1.7-fold) than similar cDNA clones and these foci were more readily cloned (3.6-fold) into permanent cell lines. A series of deletion mutants of the genomic p53 clone were employed to show that the presence of intron 4 in the p53 gene was sufficient to provide much enhanced clonability of transformed foci from culture dishes. The presence of introns in the p53 gene constructions also resulted in elevated levels of p53 protein in the p53-plus-ras-transformed cell lines. Thus, qualitative changes in the p53 protein are required to activate p53 for transformation with the oncogene ras. Quantitative improvements of transformation frequencies are associated with the higher expression levels of altered p53 protein that are provided by having one of the p53 introns in the transforming plasmid.