Activation of N-ras in a human melanoma cell line.
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.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D005347:Fibroblasts; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics; D009857:Oncogenes; D016283:Proto-Oncogene Proteins p21(ras); D014162:Transfection
D009363:Neoplasm Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)