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Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli
Abstract:
Amplification of cellular proto-oncogenes has been implicated in the development of human malignancies. A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene. Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras. Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras). The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species. A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42. It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E. coli.
Insights
Researchers identified a novel amplified ras gene in lung tumors, distinct from known ras genes. This gene, located on chromosome 12, impacts DNA topoisomerase activity in bacteria.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Proto-oncogene amplification is linked to human cancer development.
- A lung tumor with amplified c-Ki-ras 2 gene was used for study.
Purpose of the Study:
- To isolate and characterize novel amplified ras genes in human malignancies.
- To investigate the chromosomal location and evolutionary conservation of the amplified gene.
- To explore potential functional implications of the amplified gene.
Main Methods:
- Genomic DNA library construction from a lung tumor.
- Hybridization techniques using v-Ki-ras probe for gene isolation.
- Human-hamster hybrid cell studies for gene mapping.
- Restriction mapping and DNA sequencing for gene characterization.
- Plasmid subclone analysis for functional studies.
Main Results:
- A novel ras-homologous fragment was isolated and found amplified in the tumor DNA.
- The gene is located on human chromosome 12, similar to c-Ki-ras.
- The isolated fragment has a distinct restriction map compared to known Ha, Ki, and N-ras genes.
- Sequence analysis revealed evolutionary conservation across mammalian species.
- A subclone (pK42) exhibited supercoil heterogeneity, suggesting an effect on DNA topoisomerase activity in E. coli.
Conclusions:
- A novel amplified ras gene, distinct from known ras oncogenes, is identified in lung tumors.
- The gene's localization to chromosome 12 and evolutionary conservation suggest its biological significance.
- The findings indicate a potential role in oncogenesis and suggest interaction with DNA topoisomerase.