PURIFICATION OF A MOUSE NUCLEAR FACTOR THAT BINDS TO BOTH THE A AND B CORES OF THE POLYOMAVIRUS ENHANCER
PURIFICATION OF A MOUSE NUCLEAR FACTOR THAT BINDS TO BOTH THE A AND B CORES OF THE POLYOMAVIRUS ENHANCER
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DOI：
10.1128/jvi.64.10.4808-4819.1990
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发表时间：
1990-10-01
影响因子：
5.4
通讯作者：
SHIGESADA, K
中科院分区：
文献类型：
--
作者：
KAMACHI, Y;OGAWA, E;SHIGESADA, K
我们以前已经确定了一个蛋白质因子，PEBP 2（多瘤病毒增强子结合蛋白），在小鼠NIH 3 T3细胞的核提取物中，它结合到序列基序，PEA 2，位于多瘤病毒增强子A元件。在用活化的癌基因c-Ha-ras进行细胞转化时，该因子经常经历剧烈的分子修饰，变成具有显著减小的分子大小的改变形式。在这项研究中，改变的形式，PEBP 3，被纯化到接近同质。纯化的PEBP 3包含两组多肽家族，α- 1至α- 4和β- 1至β- 2，其大小分别为30至35千道尔顿和20至25千道尔顿。两种多肽的DNA结合活性具有完全相同的序列特异性。个体α或β与DNA复合的多肽显示出比PEBP 3更快的凝胶迁移率。然而，当α.和β将多肽以任意对混合在一起。这些观察结果沿着UV-和化学-交联研究的结果，使我们得出结论，PEBP 3是α的异二聚体。和β亚基，潜在地具有二价DNA结合活性。此外，发现PEBP 3结合多瘤病毒增强子的第二个迄今未被注意的位点，该位点位于B元件内，并且与先前称为猿猴病毒40增强子核心同源性的序列一致。通过比较该结合位点和原始结合位点，PEBP 3的共有序列被定义为PuACCPuCA。这些发现为PEBP 3和PEBP 2的生物学意义提供了新的见解。
We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, .alpha.-1 to .alpha.-4 and .beta.-1 to .beta.-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides DNA-binding activities with exactly the same sequence specificity. Individual .alpha. or .beta. polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when .alpha. and .beta. polypeptides were mixed together in any arbitrary pair. These observations along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of .alpha. and .beta. subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.