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Unusual chromosome structure of fission yeast DNA in mouse cells
J McManus1, P Perry, A T Sumner
1MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland.
Abstract:
Chromosomes from the fission yeast Schizosaccharomyces pombe have been introduced into mouse cells by protoplast fusion. In most cell lines the yeast DNA integrates into a single site within a mouse chromosome and results in striking chromosome morphology at metaphase. Both light and electron microscopy show that the yeast chromosome region is narrower than the flanking mouse DNA. Regions of the yeast insert stain less intensely with propidium iodide than surrounding DNA and bear a morphological resemblance to fragile sites. We investigate the composition of the yeast transgenomes and the modification and chromatin structure of this yeast DNA in mouse cells. We suggest that the underlying basis for the structure we see lies above the level of DNA modification and nucleosome assembly, and may reflect the attachment of the yeast DNA to the rodent cell nucleoskeleton. The yeast integrant replicates late in S phase at a time when G bands of the mouse chromosomes are being replicated, and participates in sister chromatid exchanges at a high frequency. We discuss the implications of these studies to the understanding of how chromatin folding relates to metaphase chromosome morphology and how large stretches of foreign DNA behave when introduced into mammalian cells.
Insights
Foreign yeast chromosomes integrated into mouse cells created narrower DNA regions, resembling fragile sites. This suggests yeast DNA attaches to the rodent cell
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Introduction of foreign DNA into mammalian cells can alter host cell chromosome structure and behavior.
- Understanding the integration and structural consequences of large DNA inserts is crucial for gene therapy and synthetic biology applications.
Purpose of the Study:
- To investigate the structural and functional characteristics of integrated yeast chromosomes in mouse cells.
- To explore the chromatin structure and replication timing of foreign DNA within a mammalian genome.
Main Methods:
- Protoplast fusion was used to introduce Schizosaccharomyces pombe chromosomes into mouse cell lines.
- Light and electron microscopy were employed to analyze chromosome morphology.
- Propidium iodide staining was used to assess DNA staining intensity and chromatin structure.
Main Results:
- Yeast DNA integrated into a single site within mouse chromosomes, creating a morphologically distinct, narrower region.
- The integrated yeast DNA regions stained less intensely with propidium iodide, resembling fragile sites.
- The yeast integrant replicated late in S phase and exhibited high-frequency sister chromatid exchanges.
Conclusions:
- The observed structure of integrated yeast DNA suggests a basis beyond DNA modification or nucleosome assembly, potentially involving attachment to the rodent cell nucleoskeleton.
- Foreign DNA integration can significantly impact host chromosome morphology and behavior.
- These findings offer insights into chromatin folding, metaphase chromosome structure, and the behavior of large foreign DNA segments in mammalian cells.