How a replication origin and matrix attachment region accelerate gene amplification under replication stress in mammalian cells
| Autor: | Noriaki Shimizu, Shun-suke Tanaka, Sho-hei Mitsuda |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
DNA Replication
Sequence analysis lcsh:Medicine Bioengineering Replication Origin Context (language use) CHO Cells Biology Origin of replication Cytogenetics Cricetulus Plasmid Stress Physiological Cricetinae Gene duplication Dihydrofolate reductase Genetics Cancer Genetics Animals Scaffold/matrix attachment region lcsh:Science Multidisciplinary Biology and life sciences Chromosome Biology Chromosomal fragile site lcsh:R Gene Amplification Cell Biology DNA Matrix Attachment Regions Molecular biology Tetrahydrofolate Dehydrogenase biology.protein lcsh:Q Research Article Biotechnology |
| Zdroj: | PLoS ONE, Vol 9, Iss 7, p e103439 (2014) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer. |
| Databáze: | OpenAIRE |
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