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In the authors' previous investigations, they established that the tumorous cell lines SK-BR-3 (breast cancer), LAN-1 (neuroblastoma), and a heterotransplant of malignant melanoma Jal contain transforming genes of Ha-ras type. Now, the authors report their results using restriction endonucleases of MspI and HpaII restriction to study nucleotide sequences 5'-CCGGC-3' and 3'GGCCG-5', which contain the 12th codon of GGC for the amino acid glycine in the normal allele of c-Ha-rasl in the three tumors listed above, in addition to human adenocarcinoma of the stomach (CaVSt) and normal cells corresponding to them. For hybridization of MspI/HpaII, fragments of chromosomal DNA isolated from cell lines SK-BR-3, and LAN-1, Ja-1 heterotransplant, and stomach adenocarcinoma CaVSt, the XmaI section of EJ oncogene, c-Ha-rasl (plasmid pEJ 6.6), labeled with ³²P was used in down-translation reaction. Hybridization was performed in 3 x SSC buffer containing 5x Deinhardt's reagent and 10% dextran sulfate at 68°C for 16-18 h. Washing of filters was conducted under rigid conditions. For autoradiography, Kodak XR-5 x-ray film in cartridges with reinforcing shields was used at -70°C, exposure time of four to six days.
 
Top-cited authors
Alexey Olovnikov
  • Russian Academy of Sciences
G. F. Krymsky
  • Institute of Computational Mathematics and Mathematical Geophysics SB RAS
B. Dubrovin
  • Scuola Internazionale Superiore di Studi Avanzati di Trieste
Anatoly Vershik
  • Russian Academy of Sciences
LP Shilnikov