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Updated: May 9, 2026
08:06
Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement
Published on: January 19, 2017
Murine genomic DNA sequences replicating autonomously in mouse L cells.
1Institute of Biochemistry, University of Würzburg, Federal Republic of Germany.
Cell
|February 12, 1988
Summary
Researchers identified novel autonomously replicating sequences (ARS) in mouse cells. These ARS elements are crucial for DNA replication and may contain conserved regulatory sequences.
Area of Science:
- Molecular Biology
- Genetics
- Mammalian Cell Culture
Background:
- Autonomous replication is essential for maintaining extrachromosomal DNA elements in eukaryotic cells.
- Identifying sequences that confer autonomous replication is key to understanding DNA replication origins and developing gene delivery vectors.
Purpose of the Study:
- To isolate and characterize DNA fragments from mouse Ltk- cells that enable autonomous replication in these cells.
- To investigate the properties and potential conserved elements within these newly identified autonomously replicating sequences (ARS).
Main Methods:
- Construction of recombinant plasmids containing random genomic DNA fragments and a selectable marker (HAT resistance).
- Transformation of mouse L cells and selection for HAT resistance.
- Confirmation of plasmid presence and replication using DNA hybridization, electron microscopy, and bacterial transformation.
- Characterization of isolated ARS using restriction enzyme analysis, bromodeoxyuridine substitution, copy number determination, and segregation analysis.
Main Results:
- Successfully isolated nineteen distinct DNA fragments conferring autonomous replication in mouse L cells.
- Demonstrated the presence and independent replication of these plasmids in transformed cells.
- Sequence analysis of nine ARS revealed a conserved 12 bp element (CTCATGAGAGGCCAA) in five of them.
Conclusions:
- Identified novel murine autonomously replicating sequences (ARS) with the potential to function as replication origins.
- The conserved 12 bp element may play a role in the initiation or regulation of DNA replication.
- These findings contribute to the understanding of DNA replication mechanisms in mammalian cells.
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