Oda K, Kato H, Saito I, Sugano S, Maruyama K, Masuda M, Shiroki K, Shimojo H
J Virol. 1983 Jan;45(1):408-19. doi: 10.1128/JVI.45.1.408-419.1983.
Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.
构建了携带腺病毒12型E1A基因的猿猴病毒40（SV40）重组体。通过去除大部分VP1基因和晚期16S RNA间隔序列的内部部分，并将5'和3'剪接位点连接成一个小片段，构建了SV40表达载体。将带有或不带有自身启动子的腺病毒12型E1A基因插入到SV40晚期启动子和剪接连接点的下游。通过与早期温度敏感突变体（tsA58）DNA共转染作为辅助，在猴细胞中繁殖和包装重组DNA。对感染所得病毒株的猴细胞进行免疫荧光染色显示，高达20%的细胞在细胞核中过量产生E1A基因产物。产物的二维凝胶电泳表明，这些产物与腺病毒12型感染的人胚肾细胞中合成的真实多肽非常相似或相同。无论E1A启动子是否存在，E1A mRNA均从SV40晚期启动子起始，并在E1A或SV40聚腺苷酸化信号处终止。这些杂合mRNA在E1A编码区被正确剪接。
