Upcroft P, Skolnik H, Upcroft J A, Solomon D, Khoury G, Hamer D H, Fareed G C
Proc Natl Acad Sci U S A. 1978 May;75(5):2117-21. doi: 10.1073/pnas.75.5.2117.
The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.
本文描述了将大肠杆菌基因导入哺乳动物细胞的过程。在体外将一个抑制性tRNA基因与猿猴病毒40(SV40)载体相连,然后用重组体转染大鼠胚胎细胞和猴肾细胞。杂交的SV40基因组,即SV40-su+III,保留了自主复制和细胞转化所需的遗传信息,并且在晚期基因区域(在SV40基因图谱上位于0.735的限制性内切酶位点Hpa II和0/1.0的EcoRI之间)有一个1300碱基对的DNA片段被一个含有抑制性tRNA基因su+III(tRNATyrsu+III)的870碱基对细菌DNA片段所取代。通过DNA重缔合动力学分析以及对来自转化的大鼠胚胎细胞和持续感染的猴细胞的总细胞DNA进行限制性酶切,确定了SV40-su+III嵌合体的结构和命运。用对载体(SV40)或su+III DNA序列特异的放射性标记探针进行杂交,结果显示主要是未整合的或游离的杂交基因组。在这两种细胞类型的克隆系中,通过限制性内切酶切下的片段长度以及其与放射性标记的细菌DNA探针而非SV40探针杂交的能力判断,细菌DNA片段完整回收。