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The silent P mating type locus in fission yeast contains two autonomously replicating sequences
Abstract:
We show that in fission yeast two DNA fragments at the silent P mating type locus provide plasmids with the capability of autonomous replication. Bacterial vectors containing these sequences replicate in a polymeric form in fission yeast very much like plasmids with the commonly used replication sequence ars1, do. There are, however, several differences between the two new ars sequences. The percentage of cells containing the plasmid during selection, the plasmid copy number and the plasmid segregation during mitosis are all dependent on the choice of the ars sequence. A DNA fragment with ars activity from the left side of the silent P cassette represses the expression of the marker gene, ura4+, at least three hundred fold compared to plasmids containing only the other new ars sequence or only ars1. The importance of replication in this promoter independent transcriptional regulation is further substantiated by the fact that the repression is partially released in the presence of ars1 on the same plasmid.
Insights
Two DNA fragments from fission yeast enable autonomous replication of plasmids. These sequences influence plasmid stability and gene expression, offering new tools for yeast genetics research.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Epigenetics
Background:
- Autonomous replication sequences (ARS) are crucial for plasmid maintenance in yeast.
- The silent P mating type locus in fission yeast is a target for studying DNA replication and epigenetic regulation.
- Existing ARS sequences like ars1 are widely used but have limitations.
Purpose of the Study:
- To identify and characterize novel ARS sequences from the silent P mating type locus in fission yeast.
- To investigate the impact of these new ARS sequences on plasmid replication, copy number, and segregation.
- To explore the role of these ARS sequences in regulating gene expression, particularly in a promoter-independent manner.
Main Methods:
- Cloning of DNA fragments from the silent P mating type locus into bacterial vectors.
- Transformation of fission yeast with engineered plasmids and selection for autonomous replication.
- Analysis of plasmid replication efficiency, copy number, and mitotic segregation.
- Quantitative assessment of marker gene (ura4+) expression in the presence of different ARS sequences.
Main Results:
- Two DNA fragments from the silent P mating type locus confer autonomous replication capabilities to plasmids in fission yeast.
- These novel ARS sequences exhibit distinct characteristics in terms of plasmid stability and copy number compared to ars1.
- A specific ARS fragment from the left side of the silent P cassette significantly represses ura4+ expression, indicating a role in transcriptional regulation independent of promoter activity.
- This repression is partially alleviated when ars1 is present on the same plasmid, suggesting an interplay between replication and gene silencing.
Conclusions:
- Novel ARS sequences from the silent P mating type locus provide versatile tools for plasmid replication in fission yeast.
- These sequences possess unique properties influencing plasmid dynamics and gene expression regulation.
- The findings highlight the importance of replication origins in promoter-independent transcriptional control and offer new avenues for genetic manipulation in yeast.