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The mcm2 mutation of yeast affects replication, rather than segregation or amplification of the two micron plasmid
Abstract:
We have studied the maintenance of the endogenous two micron (2 mu) plasmid in a strain of yeast carrying the nuclear mutation mcm2. This mutation, earlier shown to affect the maintenance of yeast minichromosomes in an ARS-dependent manner, also affected the copy number of the 2 mu plasmid. The effect was more pronounced at 35 degrees C leading to the elimination of the plasmid from the cells cultured at this temperature. The mutant cells could be efficiently cured of the circle by transformation with 2 mu ORI-carrying hybrid vectors, an observation consistent with the low copy number of the endogenous plasmid. A chromosomal revertant of this mutant for another ARS(ARS1) was found also to confer stability on the 2 mu ORI-carrying minichromosomes and had elevated levels of the endogenous plasmid. The mutation neither affected the segregation nor the amplification process mediated by site-specific recombination at FRT sites requiring the FLP gene-encoded protein action. ARS131C, an ARS that was unaffected in the mutant at 25 degrees C, could elevate the copy number of a 2 mu hybrid vector in the mutant cells. In view of these results, some aspects of segregation and copy number control of the endogeneous plasmid have been discussed. We propose that the mutation impairs the 2 mu ORI function, leading to its loss.
Insights
The mcm2 mutation in yeast disrupts the maintenance of the two micron (2 mu) plasmid, reducing its copy number and causing elimination at higher temperatures. This suggests the mutation impairs the 2 mu origin of replication function.
Area of Science:
- Yeast genetics
- Molecular biology
- Plasmid maintenance
Background:
- The endogenous 2-micron (2 mu) plasmid is a high-copy number element in yeast, crucial for understanding plasmid stability.
- Nuclear mutations affecting minichromosome maintenance can provide insights into plasmid replication and segregation.
- The mcm2 mutation is known to impact ARS-dependent minichromosome stability.
Purpose of the Study:
- To investigate the effect of the mcm2 nuclear mutation on the maintenance and copy number of the endogenous 2 mu plasmid in yeast.
- To elucidate the role of the mcm2 mutation in plasmid stability and replication.
- To understand the underlying mechanisms of 2 mu plasmid copy number control.
Main Methods:
- Yeast strain construction with the mcm2 mutation.
- Culturing mutant yeast strains at different temperatures (25°C and 35°C).
- Assessing 2 mu plasmid copy number and stability using transformation with 2 mu ORI-carrying hybrid vectors.
- Analyzing segregation and amplification mediated by FLP recombinase and FRT sites.
- Investigating the effect of ARS elements on plasmid maintenance in mutant cells.
Main Results:
- The mcm2 mutation significantly reduced the copy number of the endogenous 2 mu plasmid.
- Plasmid elimination was observed in mcm2 mutant cells cultured at 35°C.
- Mutant cells were efficiently cured of the 2 mu plasmid by transformation, indicating low copy number.
- A chromosomal revertant restored 2 mu plasmid stability and elevated copy number.
- The mutation did not affect FLP-mediated recombination or amplification.
- ARS131C partially restored copy number of a 2 mu hybrid vector in mutant cells.
Conclusions:
- The mcm2 mutation impairs the maintenance and copy number control of the endogenous 2 mu plasmid.
- The mutation likely interferes with the 2 mu origin of replication (ORI) function, leading to plasmid loss.
- These findings contribute to understanding the mechanisms of plasmid segregation and copy number regulation in yeast.