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Stable episomal maintenance of yeast artificial chromosomes in human cells
K Simpson1, A McGuigan, C Huxley
1Imperial College School of Medicine at St. Mary's London, United Kingdom.
Abstract:
Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.
Insights
The Epstein-Barr virus origin of plasmid replication (oriP) domain enables stable maintenance of large yeast artificial chromosomes (YACs) as episomes in human cells. This oriP-EBNA-1 interaction ensures YAC stability and facilitates gene expression studies.
Area of Science:
- Molecular Biology
- Epigenetics
- Mammalian Cell Culture
Background:
- Plasmids with Epstein-Barr virus origin of plasmid replication (oriP) replicate autonomously in human cells.
- Yeast artificial chromosomes (YACs) are large DNA molecules that can be manipulated in yeast.
Purpose of the Study:
- To investigate the stable episomal maintenance of large yeast artificial chromosomes (YACs) in human cells using the Epstein-Barr virus origin of plasmid replication (oriP) domain.
- To assess the stability and integrity of oriP-containing YACs (OriPYACs) over time and in the absence of selection.
Main Methods:
- Circularization of YACs in yeast via homologous recombination.
- Introduction of the oriP domain into YACs.
- Maintenance of OriPYACs in human cells expressing the Epstein-Barr virus protein EBNA-1.
- Analysis of OriPYAC integrity and stability using fluorescence in situ hybridization (FISH).
Main Results:
- The oriP-EBNA-1 interaction was sufficient for stable episomal maintenance of YACs up to 660 kb.
- A 90-kb OriPYAC remained unrearranged in all analyzed cell lines.
- A 660-kb OriPYAC was maintained intact in two out of three cell lines.
- OriPYACs showed relative stability even without selection and were associated with host cell chromosomes.
Conclusions:
- The oriP-EBNA-1 system provides stable episomal maintenance for large DNA molecules (at least 660 kb) in human cells.
- OriPYACs do not undergo significant rearrangements over time, suggesting a mechanism for stable replication.
- The association of OriPYACs with host chromosomes implies cell cycle-dependent replication and stability, similar to the viral genome.
- OriPYACs are valuable tools for studying gene function and gene expression control due to their stability and ease of manipulation.