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Characterization of the integration site of the CMV mtr in a tumor cell line
Abstract:
Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.
Insights
A human cytomegalovirus (CMV) DNA fragment transformed rodent cells, leading to tumor formation in mice. This study details the integration and rearrangement of viral DNA within host cells.
Area of Science:
- Molecular Biology
- Virology
- Oncology
Background:
- A specific fragment of human cytomegalovirus (CMV) DNA, CMV mtr, has previously demonstrated the ability to morphologically transform rodent cells in vitro.
- Understanding the mechanisms of viral oncogenesis and host-cell interactions is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the in vitro transformation potential of a human cytomegalovirus (CMV) DNA fragment.
- To characterize the molecular mechanisms underlying viral DNA integration and its role in cellular transformation and tumor development.
Main Methods:
- Co-transformation of NIH3T3 cells with CMV DNA and a G418 resistance plasmid.
- Dot blot and Southern blot hybridization for viral DNA detection and integration analysis.
- Construction of a bacteriophage lambda library for isolating integrated viral and flanking cellular DNA sequences.
- DNA sequence analysis to determine integration sites and identify rearrangements.
Main Results:
- Approximately 30% of transformed NIH3T3 cell lines retained CMV sequences.
- Selected cell lines exhibited anchorage-independent growth and formed tumors in nude mice, indicating oncogenic potential.
- Analysis revealed complex rearrangements between the viral and resistance plasmids upon integration.
- Integration occurred within repetitive mouse DNA sequences, with adjacent cellular sequences identified.
Conclusions:
- The CMV DNA fragment possesses oncogenic properties, capable of inducing cellular transformation and tumor formation.
- Viral DNA integration into host genomes can involve complex rearrangements and occur within repetitive DNA elements.
- Further investigation into these integration events can elucidate viral oncogenesis mechanisms.