Sequence organization and expression of a yeast plasmid DNA

Area of Science:

• Molecular Biology
• Yeast Genetics
• Plasmids

Background:

• Saccharomyces cerevisiae strain A364A D5 harbors 6230 bp circular double-stranded DNA molecules, known as 2-micron DNA (2µm DNA).
• These 2µm DNA molecules are present in approximately 68 copies per cell, constituting 2.4% of the haploid genome.
• A small fraction (0.4%) of non-polyadenylated yeast RNA hybridizes to these DNA circles.

Purpose of the Study:

• To characterize the structure and populations of 2µm DNA in Saccharomyces cerevisiae.
• To investigate the role of inverted repeat sequences in 2µm DNA organization.
• To analyze the expression of genes encoded by 2µm DNA when cloned in E. coli.

Main Methods:

• Electron microscopy was used to visualize denatured and self-annealed 2µm DNA, revealing a characteristic "dumbbell" shape.
• Eco-RI digestion and agarose gel electrophoresis were employed to analyze the fragments of purified 2µm DNA.
• Cloning of 2µm DNA into Escherichia coli using the pSC101 plasmid facilitated separation and analysis of different circle populations.
• Heteroduplex analysis was performed on cloned circles to study intramolecular recombination.
• Polypeptide synthesis in bacterial mini-cells containing hybrid plasmids was analyzed using acrylamide gel electrophoresis.

Main Results:

• The "dumbbell" shape observed via electron microscopy indicated the presence of a 630 bp non-tandem inverted repeat sequence within each 2µm DNA circle.
• Eco-RI digestion yielded four fragments, suggesting the existence of two distinct populations of 2µm DNA circles with the same molecular weight.
• Cloning and heteroduplex analysis confirmed that intramolecular recombination between the inverted repeats generates these two populations.
• Analysis of polypeptide synthesis revealed distinct protein patterns from hybrid plasmids containing 2µm DNA compared to the pSC101 plasmid alone.

Conclusions:

• The 2µm DNA in Saccharomyces cerevisiae exists in at least two populations due to intramolecular recombination within its inverted repeat sequences.
• These findings provide insights into the structural dynamics and potential functional implications of 2µm DNA in yeast.
• The study demonstrates the utility of cloning and heteroduplex analysis for characterizing complex plasmid structures and recombination events.