Miki T, Fleming T P, Crescenzi M, Molloy C J, Blam S B, Reynolds S H, Aaronson S A
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5167-71. doi: 10.1073/pnas.88.12.5167.
We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest.
我们开发了一种表达性cDNA克隆系统，该系统能够生成具有单向插入cDNA片段的高复杂性文库，并允许进行高效的质粒拯救。作为该系统的一个应用，从由小鼠肝细胞癌DNA诱导的NIH 3T3转化体构建了一个cDNA文库。用文库DNA转染NIH 3T3细胞导致检测到几个转化灶，从中可以拯救出具有转化能力的相同质粒。对cDNA克隆的结构和序列分析表明，该癌基因是由涉及一个未知基因和B-raf原癌基因小鼠同源物的重组事件产生的。在独立的原发性转化体中检测到相同的基因重排，这意味着癌基因的产生发生在肿瘤内，而不是在DNA转染或cDNA文库构建过程中。所鉴定克隆的高频率以及所分离的一些转化性cDNA插入片段的大尺寸表明，这种哺乳动物表达克隆系统在分离具有生物学意义的cDNA方面具有广泛的适用性。
