Rapp U R, Goldsborough M D, Mark G E, Bonner T I, Groffen J, Reynolds F H, Stephenson J R
Proc Natl Acad Sci U S A. 1983 Jul;80(14):4218-22. doi: 10.1073/pnas.80.14.4218.
We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.
我们已通过分子克隆技术获得了一种独特的急性转化性复制缺陷型小鼠C型病毒（3611 - MSV），并对其获得的癌基因进行了表征。该病毒基因组与莫洛尼（M）小鼠白血病病毒（MuLV）极为相似，只是在p30中部和聚合酶基因（pol）中部的M - MuLV序列中有一个替换。异源双链分析表明，M - MuLV DNA的2.4千碱基被1.2千碱基的细胞DNA所取代。通过DNA序列分析确定病毒与细胞序列之间的连接点位于p30序列的517个核苷酸处以及聚合酶序列的1920个核苷酸处。通过直接杂交或比较其细胞同源物的限制性片段，将3611 - MSV的转化基因（命名为v - raf）与先前分离的逆转录病毒癌基因进行比较，结果显示它是独特的。用克隆的3611 - MSV前病毒DNA转染NIH 3T3细胞可导致高效转化，回收的病毒在小鼠体内引发典型的3611 - MSV病毒肿瘤。转染的NIH 3T3细胞表达两种3611 - MSV特异性多聚蛋白（P75和P90），二者均含有与获得序列（v - raf）翻译产物相连的NH2末端gag基因编码成分。细胞同源物c - raf在小鼠和人类DNA的单倍体基因组中以一或两个拷贝存在。
