Padua R A, Barrass N C, Currie G A
Mol Cell Biol. 1985 Mar;5(3):582-5. doi: 10.1128/mcb.5.3.582-585.1985.
DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.
从人黑色素瘤细胞系Mel Swift分离得到的DNA可转化NIH3T3细胞。对二代集落的DNA进行Southern印迹分析，发现了与人类重复序列克隆blur 8杂交的8.8千碱基和7.8千碱基的保守EcoRI片段。激活的转化基因被鉴定为N-ras，并克隆了来自二代转化体的8.8千碱基EcoRI片段。使用跨越正常密码子61（CAA）或突变密码子61（AAA）的合成17聚体寡核苷酸进行杂交。来自黑色素瘤细胞系Mel Swift的克隆N-ras与突变（AAA）寡核苷酸杂交。由此我们预测氨基酸61处谷氨酰胺被赖氨酸取代，这一变化通过对细胞系Mel Swift的N-ras第一和第二外显子进行常规测序得到证实。转染实验表明，只有那些第61位发生突变的重组克隆具有生物学活性。
