Use of the two-hybrid system to identify the domain of p53 involved in oligomerization.
Use of the two-hybrid system to identify the domain of p53 involved in oligomerization.
复制标题
DOI：
--
复制
发表时间：
1993-06
期刊：
影响因子：
8
通讯作者：
Kuniyoshi Iwabuchi;Bin Li;P. Bartel;S. Fields
中科院分区：
文献类型：
--
作者：
Kuniyoshi Iwabuchi;Bin Li;P. Bartel;S. Fields
我们使用了一种基于酵母的遗传检测方法——双杂交系统，来鉴定肿瘤抑制因子p53参与寡聚化的结构域。该检测方法依赖于转录激活因子酵母GAL4蛋白功能的重建，通过与GAL4的DNA结合结构域融合的蛋白质和与GAL4的转录激活结构域融合的蛋白质之间的相互作用来实现。我们通过一个重建实验表明，这种方法能够检测缺失其N端激活结构域的p53与SV40大T抗原的相互作用。然后，我们搜索了一个以激活结构域融合形式存在的人类蛋白质文库，以寻找能够与p53和DNA结合结构域的融合蛋白相互作用的蛋白质。这次搜索鉴定出了36个含有p53基因的质粒，代表了10个不同的类别。这些结果为p53的寡聚化提供了额外的体内证据。从文库筛选中鉴定出的最小的p53片段仅包含氨基酸331 - 393，这表明这个小的C端片段足以介导寡聚化。此外，在该检测中，一种突变的p53蛋白能够与野生型蛋白结合，这为突变形式的p53通过与野生型的C端寡聚化以显性负性方式起作用的观点提供了支持。
We used a yeast-based genetic assay, the two-hybrid system, to characterize the domain of the tumor-suppressor p53 involved in oligomerization. This assay relies on the reconstitution of the function of a transcriptional activator, the yeast GAL4 protein, via the interaction of a protein fused to the DNA-binding domain of GAL4 with a protein fused to the transcriptional activation domain of GAL4. We show by a reconstruction experiment that this approach could detect the interaction of p53 deleted for its N-terminal activation domain with SV40 large T antigen. We then searched a library of human proteins present as activation domain hybrids for proteins that can interact with the hybrid of p53 with the DNA-binding domain. This search identified 36 plasmids containing the p53 gene, representing 10 different classes. These results provide an additional in vivo demonstration of p53 oligomerization. The smallest p53 fragment identified from screening the library contained only amino acids 331-393, indicating that this small C-terminal fragment is sufficient to mediate oligomerization. In addition, a mutant p53 protein could bind to the wild-type protein in this assay, providing support for the idea that mutant forms of p53 act in a dominant-negative manner through C-terminal oligomerization with the wild type.