Lassam N J, Bayley S T, Graham F L
Cell. 1979 Nov;18(3):781-91. doi: 10.1016/0092-8674(79)90131-4.
We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.
我们通过从感染野生型（wt）Ad5或转化缺陷型宿主范围（hr）突变体的细胞以及被Ad5转化的细胞中免疫沉淀抗原（使用双抗体和蛋白A-琼脂糖技术），研究了与Ad5基因组转化区表达相关的多肽。使用了三种不同的抗血清：对早期病毒产物特异的P抗血清（Russell等人，1967年）以及两种不同的仓鼠肿瘤抗血清。对感染wt的KB细胞的抗原进行免疫沉淀，随后对沉淀蛋白进行SDS-聚丙烯酰胺凝胶电泳，结果显示，所有三种抗血清以及双抗体和蛋白A-琼脂糖技术均检测到一种分子量约为58,000的主要多肽，而P抗血清还沉淀出分子量分别为72,000、67,000和44,000的多肽，它们可能分别代表DNA结合蛋白和相关多肽。使用双抗体技术时，除上述蛋白质外，P抗血清和仓鼠肿瘤抗血清还沉淀出一种10,500道尔顿的多肽，而使用蛋白A-琼脂糖方法时未检测到该多肽。使用双抗体或蛋白A-琼脂糖技术，我们发现互补组II的hr突变体未能诱导58,000道尔顿蛋白的合成，而互补组I的突变体产生的量正常或接近正常。使用双抗体技术，我们发现互补组I的突变体不存在10,500道尔顿的蛋白或其产生量减少。在许多不同的Ad5转化细胞系中检测到一种58,000道尔顿的蛋白，包括293人细胞系、14b仓鼠细胞系和几种转化的大鼠细胞系。这一观察结果以及转化阴性的互补组II突变体未能诱导58,000道尔顿多肽合成这一事实表明，该蛋白是细胞转化所必需的Ad5特异性产物之一。
