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Yeast episomal plasmids (YEps) are shuttle vectors. They can replicate in E. coli and also in yeast. The yeast episomal plasmids and has the following parts: 1. The 2 Micron (µm) Plasmid, 2. LEU2 Gene, and 3. The pBR 322.
Part # 1. The 2 Micron (µm) Plasmid:
This plasmid is found in several strains of yeast, Saccharomyces cerevisiae. It is 6 kb in size and has a copy number between 70 and 200. It replicates autonomously by using several enzymes provided by the host cell and the proteins coded by its own genes.
The 2µm plasmid has an origin of replication and two genes involved in replication, and also encodes a site-specific recombination protein, called FLP, which is homologous to the phage λ, integrase and can invert part of the 2µm DNA sequence.
Part # 2. LEU2 Gene:
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LEU2 gene is a gene of yeast chromosome. It codes for an enzyme called isopropyl malate dehydrogenase which is involved in conversion of pyruvic acid to leucine. When LEU2 gene is used as a selectable marker, the host yeast must be a mutant with a non-functional LEU2 gene. Such strains of yeast are called leu2.
These mutant cells are unable to synthesize leucine and will grow only if this amino acid is added to the growth medium. However, yeast cells transformed by the YEp (yeast episomal plasmid) containing LEU2 gene can grow in the growth medium lacking the amino acid leucine. Thus, the transformed yeast cells (called transformants) can be selected.
Part # 3. The pBR 322:
It is the bacterial plasmid. It has two marker genes: the ampicillin and tetracycline resistant genes (i.e., AmpR and TetR). This plasmid has its own origin of replication. Hence, initial gene manipulation experiments can be done with E. coli.
The YEP vector is episomal in nature as it can integrate with one of the yeast chromosomes. Integration occurs by homologous recombination between the LEU2 gene. This results in the insertion of the entire YEP into on of the yeast chromosome.
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The plasmid may remain integration or it may excise later. For cloning a particular gene in yeast, the initial cloning experiment is done with E. coli. The recombinants are multiplied and selected. The recombinant plasmids are then purified and introduced into yeast where the new gene will express itself.
