Ishikawa F, Sakai R, Ochiai M, Sugimura T, Nagao M
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Nucleic Acids Symp Ser. 1988(19):39-42.
Activation mechanism of raf oncogene was studied by applying in vitro mutagenesis to its cDNA. Previous studies suggested the presence of an activation suppressing sequence in the amino-terminal half of c-raf product. Loss of the sequence by genetic rearrangement was presumed to convert c-raf to possess transforming activity. To identify such sequence, we prepared cDNA mutants by random linker insertion. Synthetic oligonucleotide linker was inserted into the plasmid containing cDNA at a single and random site. Coupling two different mutants, in-frame deletion mutants were constructed systematically. Analysis of these deletion mutants revealed a region, the loss of which made c-raf activated.
通过对raf癌基因的cDNA进行体外诱变来研究其激活机制。先前的研究表明，在c-raf产物的氨基末端一半存在一个激活抑制序列。据推测，通过基因重排导致该序列缺失会使c-raf具有转化活性。为了鉴定这样的序列，我们通过随机连接子插入制备了cDNA突变体。将合成的寡核苷酸连接子随机插入到含有cDNA的质粒的单个位点。将两个不同的突变体偶联，系统地构建了读框内缺失突变体。对这些缺失突变体的分析揭示了一个区域，该区域的缺失会使c-raf被激活。
