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6.3.7 Vectors used in eukaryotes

The use ofE. colifor general cloning and manipulation of DNA is well established;

however, numerous developments have been made for cloning in eukaryotic cells.

Plasmids used for cloning DNA in eukaryotic cells require a eukaryotic origin of

replication and marker genes that will be expressed by eukaryotic cells. At present the

two most important applications of plasmids to eukaryotic cells are for cloning in

yeast and in plants.

Although yeast has a natural plasmid, called the 2mcircle, this is too large for use

in cloning. Plasmids such as the yeast episomal plasmid (YEp) have been created by

genetic manipulation using replication origins from the 2mcircle, and by incorpor-

ating a gene which will complement a defective gene in the host yeast cell. If, for

example, a strain of yeast is used which has a defective gene for the biosynthesis of an

amino acid, an active copy of that gene on a yeast plasmid can be used as a selectable

marker for the presence of that plasmid. Yeast, like bacteria, can be grown rapidly, and

it is therefore well suited for use in cloning. Of particular use has been the creation of

shuttle vectors which have origins of replication for yeast and bacteria such asE. coli.

TRP-ori-CEN

pYAC2

TEL TEL

URA3

SUP4

DNA inserted

inSmaI site

Digest with BamHI

remove DNA

fragment

Left arm (TRP-ori-CEN)

DNA to be inserted

Right arm (URA3)

Yeast Artificial Chromosome Construct

TransformationSaccharomyces cerevisiae

SmaI

BamHI BamHI

Fig. 6.24Scheme for cloning large fragments of DNA into YAC vectors.

221 6.3 Cloning vectors