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Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences
Abstract:
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.
Insights
Researchers identified a mouse oncogene similar to human N-ras. A specific mutation at amino acid 61 was found to be responsible for the gene
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Identifying oncogenes is crucial for understanding cancer development.
- Mouse models are valuable for studying human diseases like cancer.
Purpose of the Study:
- To clone and characterize a functional oncogene from mouse Lewis Lung tumor DNA.
- To investigate the genetic basis of tumor formation and identify the specific mutation responsible for oncogenic activity.
Main Methods:
- Ligation of mouse tumor DNA to a cosmid vector containing a resistance gene.
- Transfection into NIH3T3 cells followed by selection for geneticin resistance and tumorigenicity.
- Construction of cosmid libraries and isolation of kanamycin-resistant cosmids harboring the oncogene.
- Comparative analysis of the cloned oncogene with human N-ras using DNA probes.
Main Results:
- Isolation of tumorigenic NIH3T3 cells containing integrated cosmid DNA.
- Successful cloning of a functional oncogene highly related to the human N-ras gene.
- Identification of a mutation at amino acid position 61 as the cause of the rescued gene's transforming activity.
Conclusions:
- A mouse oncogene homologous to human N-ras was identified and cloned.
- A specific point mutation at position 61 confers transforming activity to the N-ras gene.
- This study provides insights into the molecular mechanisms of oncogene activation and tumorigenesis.