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Abstract:
A variety of Saccharomyces strains were examined for the presence of 2micro DNA and, if present, for the pattern of fragments produced by its digestion with site-specific (restriction) endonucleases. Two strains were found that did not contain detectable levels of 2micro DNA, and two strains contained 2micro DNA molecules having only one EcoRI restriction endonuclease recognition site rather than the usual two.-A haploid containing 2micro DNA with one EcoRI restriction site was mated with a haploid containing 2micro DNA with two EcoRI restriction sites and the resulting diploid maintained both types during vegetative growth. Sporulation of the diploid produced four spores, and the clones from these spores contained both types.-A haploid lacking 2micro DNA was mated with a haploid containing 2micro DNA and the resulting diploid contained 2micro DNA. The four clones derived from the haploid spores after sporulation of this diploid all contained 2micro DNA. A rho(-) strain without 2micro DNA was mated to a rho(+) strain with 2micro DNA, and heteroplasmons were selected that had received the nucleus from the strain without 2micro DNA and the mitochondria from the strain with 2micro DNA. Twelve of twenty-four such clones contained 2micro DNA.-I conclude that: (1) the different types of 2micro DNA identified in these strains do not restrict one another, (2) the different types are inherited extrachromosomally, (3) lack of 2micro DNA in two strains is not due to the absence of genes needed for maintenance and (4) the approximately 100 copies of 2micro DNA contained within a single cell are probably clustered within one or a few cytoplasmic organelles.
Insights
Investigating Saccharomyces strains revealed variations in 2-micron DNA, including altered restriction sites and extrachromosomal inheritance. These findings suggest 2-micron DNA is maintained by existing genes and likely clustered in organelles.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- The 2-micron DNA is a high-copy-number plasmid found in Saccharomyces cerevisiae.
- Understanding its maintenance and inheritance is crucial for yeast molecular biology.
Purpose of the Study:
- To investigate the presence and characteristics of 2-micron DNA in various Saccharomyces strains.
- To determine the inheritance patterns of different 2-micron DNA types and strains lacking it.
Main Methods:
- Screening of Saccharomyces strains for 2-micron DNA presence.
- Digestion of 2-micron DNA with EcoRI restriction endonuclease.
- Mating experiments between haploid strains with different 2-micron DNA types or absence thereof.
- Analysis of meiotic segregation and heteroplasmon formation.
Main Results:
- Two strains lacked detectable 2-micron DNA; two strains possessed variants with a single EcoRI site instead of the usual two.
- Diploid cells formed by mating strains with different 2-micron DNA types maintained both variants.
- Haploid strains lacking 2-micron DNA, when mated with strains possessing it, produced diploids and subsequent spores that contained 2-micron DNA.
- Heteroplasmons, receiving nuclei from a rho(-) strain and mitochondria from a rho(+) strain with 2-micron DNA, showed 2-micron DNA presence in half the clones.
Conclusions:
- Different 2-micron DNA types do not inhibit each other's replication or maintenance.
- 2-micron DNA is inherited extrachromosomally.
- The absence of 2-micron DNA in some strains is not due to a lack of necessary maintenance genes.
- The approximately 100 copies of 2-micron DNA per cell are likely localized within one or a few cytoplasmic organelles.