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Molecular cloning of a novel oncogene generated by DNA recombination during transfection
T Nakamura1, J Hillova, R Mariage-Samson
1Centre National de la Recherche Scientifique, Institut de Cancérologie et d'Immunogénétique, Villejuif, France.
Abstract:
Genomic DNA of a Ewing's sarcoma cell line and pKOneo were cotransfected into NIH3T3 cells and assayed for tumorigenicity in nude mice. Primary transfected tumors all contained human DNA. One transfected sequence was retained through secondary and tertiary tumors, and was cloned in four overlapping cosmids covering 51.5-kb human DNA flanked on both sides with mouse DNA. The cloned sequence, though continuous in transfected tumors, originated from three major genetic elements discontiguous in human cells and recombined during transfection. Accordingly there was one species of hybrid RNA transcripts of 9 kb in the transfected tumors and no transcripts in human cells, including Ewing's sarcoma cells. Evidence that the cloned sequence is a biologically active novel oncogene was provided by transfection of a mixture of two cosmid clones which generated tumors where they were transcribed into 9-kb RNA. The oncogene was named tre to designate its origin from transfection recombined DNA molecules.
Insights
Researchers identified a novel oncogene, named tre, formed by recombined DNA from Ewing's sarcoma cells. This oncogene drives tumor formation in mice by producing specific RNA transcripts.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ewing's sarcoma is a pediatric bone cancer.
- Understanding the genetic basis of tumorigenesis is crucial for developing targeted therapies.
Purpose of the Study:
- To identify and characterize novel oncogenes involved in Ewing's sarcoma tumorigenesis.
- To investigate the genetic recombination and expression of transfected DNA in a mouse model.
Main Methods:
- Cotransfection of Ewing's sarcoma cell line genomic DNA and pKOneo into NIH3T3 cells.
- Tumorigenicity assays in nude mice.
- DNA cloning using overlapping cosmids and RNA transcript analysis.
Main Results:
- Transfected tumors contained human DNA, with one sequence retained through multiple passages.
- A 51.5-kb human DNA sequence, recombined from three discontiguous elements, was cloned.
- This cloned sequence produced a 9-kb hybrid RNA transcript, indicating biological activity as a novel oncogene (tre).
Conclusions:
- A novel oncogene, 'tre', was identified, originating from recombined DNA elements of Ewing's sarcoma.
- The 'tre' oncogene is biologically active and drives tumor formation through the expression of a 9-kb RNA transcript.