The 2Micron Plasmid of Yeast

Plasmids are found in higher organisms, although they are less common than in bacteria. The yeast, Saccharomyces cerevisiae, has been used as a model organism for the investigation of eukaryotic molecular biology. Most strains of yeast harbor a plasmid known as the 2m circle or 2m plasmid. This is a circular molecule consisting of 6318 bp of double-stranded DNA. It is present at 50-100 copies per haploid genome and is located in the nucleus of the yeast cell, where it is bound by histones and forms nucle-osomes like chromosomal DNA. The 2m plasmid has been widely used in genetic engineering as the basis for multicopy eukaryotic cloning vectors. Similar plasmids are found in other species of yeast.

The 2m plasmid contains two perfect inverted repeats of 599 bp that separate the plasmid into two regions of 2774 and 2346 bp respectively (Fig. 16.25). The plasmid encoded Flp protein (Flp recombinase or "flippase") catalyzes recombination between

2 micron plasmid See 2m plasmid 2m circle Same as 2m plasmid

2m plasmid (or 2m circle) A multicopy plasmid found in the yeast, Saccharomyces cerevisiae, whose derivatives are widely used as vectors Flp recombinase (or flippase) Enzyme encoded by the 2m plasmid of yeast that catalyzes recombination between inverted repeats (FRT sites)

Certain DNA Molecules may Behave as Viruses or Plasmids 451

FIGURE 16.25 The 2m Plasmid of Yeast

Two alternate forms of the 2|m plasmid are inter-converted by recombination. The plasmid has two inverted repeats (IVR1 and IVR2), which can align. The enzyme, Flp recombinase, recognizes the FRT sites (flip recombination target) and makes a crossover that inverts one half of the plasmid relative to the other. Notice the top plasmid has origin (ori) close to the Rep2 sequence, whereas, the bottom plasmid has the origin on the other side (close to the FLP gene). The Rep1 and Rep2 proteins regulate both the FLP gene and the replication of the plasmid itself.

Certain DNA Molecules may Behave as Viruses or Plasmids 451

the inverted repeats. Flp recognizes a 48 bp target site (Flp recombination target, or FRT site) located within the inverted repeats. The result is the inversion of one half of the plasmid relative to the other. The two forms of the plasmid are found in roughly equal proportions. The Rep1 and Rep2 proteins regulate the expression of the FLP gene and also bind to the origin of replication (ori) and the REP3 DNA sequence.

The Flp recombinase is used in genetic engineering to control the expression of a variety of genes by inverting segments of DNA. Flp is functional in bacteria, plants and animals provided the correct recognition sites are present. In addition to the inversion reaction, Flp recombinase will promote site-specific insertion and deletion reactions of segments flanked by FRT sites. The Flp/FRT system is similar to the widely used Cre/loxP recombinase system of bacterial virus P1.

P1 combines the properties of plasmid and virus and can choose either lifestyle depending on the circumstances.

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