Dastoor M N, Shoyab M, Baluda M A
J Virol. 1977 Feb;21(2):541-7. doi: 10.1128/JVI.21.2.541-547.1977.
We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.
我们研究了禽肿瘤病毒在不同宿主基因组中的整合位点,考察了整合的前病毒DNA旁邻的细胞DNA序列的重复频率。研究了以下系统:培养的鸭胚细胞中的禽肉瘤病毒(B - 77)和禽白血病病毒(劳氏相关病毒 - 61),以及培养的小鼠3T3细胞中的B - 77。这些系统代表了宿主对病毒感染的不同反应,即一种是细胞转化和病毒复制都发生的情况(B - 77感染的鸭细胞),一种是只发生病毒复制而不发生转化的情况(劳氏相关病毒 - 61感染的鸭细胞),还有一种是只发生转化而不发生病毒复制的情况(B - 77感染的3T3细胞)。采用了两次连续杂交。首先,将大的变性DNA片段(2.8×10⁶道尔顿)复性到不同的Cot(每升摩尔秒)值。接下来,通过羟基磷灰石柱色谱法分离在不同Cot值下仍保持单链的DNA,将其固定在硝酸纤维素滤膜上,并用过量的³H标记的³⁵S病毒RNA进行杂交,以滴定前病毒DNA的浓度。结果表明,B - 77肉瘤病毒和劳氏相关病毒 - 61整合到鸭DNA的单一区域,而在转化的小鼠3T3细胞中,B - 77前病毒DNA与重复和单一的宿主DNA序列都有关联。