Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.
Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.
D000818:Animals; D002471:Cell Transformation, Neoplastic; D003360:Cosmids; D004273:DNA, Neoplasm / Q000235:genetics*; D004351:Drug Resistance; D005839:Gentamicins / Q000494:pharmacology; D008175:Lung Neoplasms / Q000235:genetics; D051379:Mice; D009374:Neoplasms, Experimental / Q000235:genetics*; D009857:Oncogenes; D014162:Transfection; D014170:Transformation, Genetic
D004273:DNA, Neoplasm; D005839:Gentamicins; C010680:antibiotic G 418