Eucaryotic cloning vectors

A number of problems are associated with the expression of complex eucaryotic genes in bacterial cloning systems, some of which have just been outlined. In order to avoid these, such genes may be expressed in eucaryotic cloning systems, the model system for which is the yeast Saccharomyces cerevisiae. This holds many attractions for the experimenter:

• it is safe and easy to handle in the laboratory

Table 12.1 Characteristics of yeast cloning vectors

Plasmid type

Abbreviation

Features

Yeast episomal plasmid

YEp

Contain 2 /m circle (naturally occurring yeast plasmid) origin of replication and whole of pBR322 sequence. Can integrate into yeast chromosomal DNA or replicate independently. High copy number

Yeast integrative plasmid

YIp

Bacterial plasmid containing yeast selectable marker. Stably integrates into yeast chromosome. Unable to exist independently in yeast. Low transformation rate and single copy number

Yeast replicative plasmid

YRp

Carries yeast chromosomal origin of replication (ARS); high copy number; unstable

Yeast centromere plasmid

YCp

Contain centromere sequence - ensures stability, but low (single) copy number

• it grows at higher density than E. coli

• it grows much faster than other eucaryotic cells in culture (e.g. cultured mammalian cells)

Several types of vector have been developed for use in yeasts. These are summarised in Table 12.1. The choice of which type to use will depend on the relative importance to a particular application of factors such as yield and long-term stability. Most yeast vectors are shuttle vectors, that is, vectors that can be replicated in more than one type of host cell (in this case, yeast and E. coli). Initial cloning is more easily done in E. coli, then the recombinant vector is transferred to yeast cells, which can carry out functions such as protein folding and glycosylation that are not possible in bacteria. Shuttle vectors must contain selectable markers and an origin of replication (or means of integration) for both cell types.

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