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Distinct mouse DNA sequences enable establishment and persistence of plasmid DNA polymers in mouse cells
Abstract:
Distinct elements isolated from mouse genomic DNA confer on plasmid DNA the ability to persist at high copy numbers in mouse L fibroblasts (1). Field inversion gel electrophoresis demonstrated that - in contrast to our previous assumption - the persisting plasmid DNA does not exist extrachromosomally but as clusters of tandem repeats integrated into genomic DNA. Digestion with restriction endonucleases that do not cut within the plasmid DNA results in fragments of 50-300 kb in length indicating reiteration of 10-50 plasmid DNA molecules. Restriction with several enzymes that cut once or twice within the plasmid sequences lead to fragment(s) indicative for head-to-tail tandem repeats. In situ hybridization revealed signals for a long homogeneously staining region (HSR) in one or two chromosomes per cell nucleus. Possibilities how these elements could act in the establishment and/or maintenance of the head-to-tail polymers of plasmid DNA in mouse cells are discussed.
Insights
Distinct DNA elements enable high-copy persistence of plasmid DNA in mouse cells. Contrary to assumptions, this DNA integrates into genomic DNA as tandem repeat clusters within chromosomes.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mammalian cells can maintain plasmid DNA at high copy numbers through specific DNA elements.
- Previous assumptions suggested extrachromosomal persistence of such plasmids.
Purpose of the Study:
- To investigate the mechanism of high-copy number plasmid DNA persistence in mouse L fibroblasts.
- To determine the genomic location and organization of persisting plasmid DNA.
Main Methods:
- Isolation of distinct DNA elements from mouse genomic DNA.
- Plasmid DNA integration studies in mouse L fibroblasts.
- Field inversion gel electrophoresis (FIGE) for analyzing DNA fragment sizes.
- Restriction endonuclease digestion to assess DNA structure.
- In situ hybridization to identify chromosomal integration sites.
Main Results:
- Persisting plasmid DNA was found integrated into genomic DNA, not extrachromosomal.
- Plasmid DNA exists as clusters of head-to-tail tandem repeats.
- FIGE revealed large fragments (50-300 kb), indicating 10-50 plasmid DNA reiterations.
- In situ hybridization localized signals to homogeneously staining regions (HSRs) on mouse chromosomes.
Conclusions:
- Specific genomic elements facilitate plasmid DNA integration and high-copy persistence.
- Plasmid DNA forms tandem arrays integrated within the host cell's genome.
- Homogeneously staining regions (HSRs) are associated with the integration of these plasmid arrays.