ASSOCIATION OF P53 BINDING AND IMMORTALIZATION OF PRIMARY C57BL/6 MOUSE EMBRYO FIBROBLASTS BY USING SIMIAN-VIRUS 40 T-ANTIGEN MUTANTS BEARING INTERNAL OVERLAPPING DELETION MUTATIONS
ASSOCIATION OF P53 BINDING AND IMMORTALIZATION OF PRIMARY C57BL/6 MOUSE EMBRYO FIBROBLASTS BY USING SIMIAN-VIRUS 40 T-ANTIGEN MUTANTS BEARING INTERNAL OVERLAPPING DELETION MUTATIONS
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DOI：
10.1128/jvi.67.4.1817-1829.1993
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发表时间：
1993-04-01
影响因子：
5.4
通讯作者：
TEVETHIA, MJ
中科院分区：
文献类型：
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作者：
KIERSTEAD, TD;TEVETHIA, MJ
为了更精确地定位T抗原的永生化和p53结合结构域，通过利用Bal 31缺失和寡核苷酸定向诱变的组合，在密码子251至651之间产生了一系列重叠缺失突变。永生化试验结果表明，氨基酸（aa）252至350，400，和451至532可以被删除，而不会严重影响永生化，虽然在某些情况下，永生化菌落的出现被延迟。Western免疫印迹实验表明，突变体缺失氨基酸252至300，301至350，400，或451至532产生的T抗原的p53结合能力相对于野生型T抗原仅略有降低。在这种缺失分析的范围内，永生化和p53结合结构域似乎是共线的，事实上，可能代表同一结构域的两个方面。这种缺失分析消除了作为p53结合或永生化过程参与者的整个锌指结构域（aa 302至320）、一小部分富含亮氨酸的区域（aa 345至350）和一大部分ATP结合结构域（aa 451至528）。结果还表明，去除区域451至532内的T抗原氨基酸似乎改变了新合成的但不是旧的T抗原和p53分子形成复合物的能力。
To more precisely map the immortalization and p53 binding domains of T antigen, a large series of overlapping deletion mutations were created between codons 251 to 651 by utilizing a combination of Bal 31 deletion and oligonucleotide-directed mutagenesis. Immortalization assay results indicated that amino acids (aa) 252 to 350, 400, and 451 to 532 could be removed without seriously compromising immortalization, although the appearance of immortal colonies was delayed in some cases. Western immunoblotting experiments indicated that the p53 binding capacities of T antigen produced by mutants missing aa 252 to 300, 301 to 350, 400, or 451 to 532 were only slightly reduced relative to that of wild-type T antigen. Within the limits of this deletion analysis, the immortalization and p53 binding domains appear to be colinear and, in fact, may represent two aspects of the same domain. This deletion analysis eliminates the entire zinc finger domain (aa 302 to 320), a small portion of the leucine-rich region (aa 345 to 350), and a large portion of the ATP binding domain (aa 451 to 528) as participants in p53 binding or in the immortalization process. The results also show that removal of T antigen amino acids within the region 451 to 532 appears to alter the capacity of newly synthesized but not older T antigen and p53 molecules to form complexes.