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Spontaneous amplification of yeast CEN ARS plasmids
Molecular & General Genetics : MGG
|July 1, 1986
Summary
Yeast plasmids with CEN4 ARS1 elements can spontaneously amplify, increasing gene copy number. This amplification leads to a His+ phenotype but results in less stable plasmids upon removal of selective pressure.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Plasmid Biology
Background:
- Transformation of Saccharomyces cerevisiae is a fundamental technique in molecular biology.
- Yeast centromere (CEN) and autonomously replicating sequence (ARS) plasmids are crucial for maintaining genetic elements.
- The his3-delta 4 allele, along with URA3 and TRP1 markers, are commonly used in yeast genetic studies.
Purpose of the Study:
- To investigate the spontaneous amplification of yeast CEN4 ARS1 plasmids.
- To understand the correlation between plasmid amplification and phenotypic changes.
- To assess the stability of amplified plasmids under varying selective pressures.
Main Methods:
- Transformation of Saccharomyces cerevisiae with CEN4 ARS1 plasmids carrying specific alleles and markers.
- Selection of transformants based on auxotrophic markers (His+, Ura+, Trp+).
- Monitoring of plasmid copy number and phenotypic stability during continuous growth and under selective pressure removal.
Main Results:
- His+ transformants appeared at lower frequencies than Ura+ Trp+ transformants.
- A His+ phenotype arose spontaneously during continuous growth, linked to increased plasmid copy number (up to 12-fold).
- The His+ phenotype and increased copy number were lost more readily than Ura+ Trp+ markers upon removal of selective pressure, indicating reduced stability of amplified copies.
Conclusions:
- Yeast CEN4 ARS1 plasmids can undergo spontaneous amplification to higher levels than previously documented.
- Amplification of these plasmids can lead to altered phenotypes but does not guarantee enhanced stability of all plasmid copies.
- The findings suggest that while amplification increases gene dosage, it may compromise the overall stability of the plasmid system in Saccharomyces cerevisiae.
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