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Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix
N Cossons1, T O Nielsen, C Dini
1McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Journal of Cellular Biochemistry
|January 24, 1998
Summary
Mammalian DNA replication origins cloned into yeast vectors stably maintained in HeLa cells. Functional matrix attachment regions (MARs) on these episomes facilitate nuclear matrix association and replication.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mammalian DNA replication initiates from specific origins.
- Episomal DNA maintenance is crucial for genetic engineering and gene therapy.
- Matrix attachment regions (MARs) are DNA elements involved in genome organization and replication.
Purpose of the Study:
- To clone mammalian DNA replication origins into a yeast vector.
- To assess the stability and episomal maintenance of these constructs in mammalian cells.
- To investigate the role of MARs in episomal DNA replication and nuclear matrix association.
Main Methods:
- Cloning of mammalian DNA replication origins (343, S3, X24) into the pYACneo yeast vector.
- Transfection of HeLa cells and isolation of stable clones.
- Long-term culture and stability assessment of episomes.
- High salt extraction of the nucleus and fluorescent in situ hybridization (FISH).
Main Results:
- Stable episomal maintenance of cloned DNA sequences in HeLa cells (C343e2 and CS3e1 lines) with ~80% stability per generation.
- Presence of functional matrix attachment regions (MARs) within the episomal sequences.
- Demonstrated association of the 343 episome with the nuclear matrix via MARs.
Conclusions:
- Functional MARs facilitate episomal DNA replication and maintenance.
- Cloned mammalian replication origins can be stably maintained as episomes in mammalian cells.
- These episomal constructs have potential applications as microchromosomes or gene delivery vectors.