el-Beik T, Razzaque A, Jariwalla R, Cihlar R L, Rosenthal L J
J Virol. 1986 Nov;60(2):645-52. doi: 10.1128/JVI.60.2.645-652.1986.
The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.
在永生化啮齿动物细胞中开展了来自人巨细胞病毒汤氏株（Towne）和AD169株的特定克隆DNA片段的转化（集落形成）活性研究。汤氏株XbaI片段E在NIH 3T3细胞中形成集落的频率比单独使用汤氏株XbaI片段AO、O、C或载体DNA时观察到的频率高80至100倍，但与来自人巨细胞病毒AD169株的转化片段pCM4127观察到的频率相似（J. A. 尼尔森、B. 弗莱肯施泰因、D. A. 加洛韦和J. K. 麦克杜格尔，《病毒学杂志》43:83 - 91, 1982年；J. A. 尼尔森、B. 弗莱肯施泰因、G. 扬、D. A. 加洛韦和J. K. 麦克杜格尔，《病毒学杂志》49:109 - 115, 1984年）。在转染汤氏株XbaI片段E的NIH 3T3细胞中，转染后5至6周首次检测到集落，而用AD169株pCM4127转染后2至3周检测到集落。用BamHI消化汤氏株XbaI片段E并没有显著降低其形成集落的活性。当分别检测汤氏株XbaI片段E的BamHI亚克隆在NIH 3T3和大鼠-2细胞中形成集落的情况时，转化活性定位于每个末端片段（EJ和EM）内。与汤氏株XbaI片段E诱导形成的集落相比，单独由EJ或EM DNA诱导形成的集落较小。分离得到的集落细胞系在软琼脂中能够生长，并且在具有免疫活性的同基因动物中具有致瘤性。用完整的EM和一个缺乏细胞相关序列的1.5千碱基亚片段通过Southern印迹杂交分析来自转化细胞系和肿瘤来源细胞系的高分子量DNA。在汤氏株XbaI片段E转化的NIH 3T3细胞中，病毒特异性EM序列在每个细胞中检测到的拷贝数少于一个，而在大鼠肿瘤来源的细胞系中检测到多个拷贝。相比之下，以完整的EJ为探针，在EJ转化和肿瘤来源的细胞系中几乎检测不到病毒特异性EJ序列。
