Big inserts, expression and shuttle vectors

Eukaryotic genes are often long and broken up by introns, so they can be too big for plasmids. For very large pieces of DNA, scientists use artificial chromosomes.

Baker's yeast, Saccharomyces cerevisiae, is the commonest eukaryotic host. It grows almost as easily as bacteria, many of its mutants are well mapped, and it is GRAS (Generally Recognised As Safe), because people have used it safely in food for thousands of years. Its natural 2 µm plasmid gave rise to several yeast vectors.

A YAC (yeast artificial chromosome) contains everything a real chromosome needs:

• two telomeres, which protect the ends;
• a centromere, which makes sure it is shared correctly at cell division;
• an ARS (autonomously replicating sequence), the yeast origin of replication;
• selectable markers.

It is kept as a circle in E. coli, and cut into a linear form for use in yeast. It can carry huge inserts, 200-500 kb. BACs (bacterial artificial chromosomes) and PACs are other high-capacity vectors.

Expression vectors do more than copy a gene: they make the host produce its protein. Around the cloning site they carry an expression cassette: a promoter (often inducible, so it can be switched on when wanted) upstream (on the 5' side), unique sites for the gene, and a terminator downstream (on the 3' side).

Making a eukaryotic protein in bacteria (heterologous expression) has limits. Bacteria cannot splice out introns, so the gene must be used without introns (for example, as cDNA copied from mRNA). And bacteria cannot make post-translational changes such as adding sugars (glycosylation), so the protein must be one that does not need them.

Shuttle vectors can replicate in two different hosts, such as E. coli and yeast. They carry two origins of replication, one for each host, but only the matching one works in a given host. This lets scientists do the easy work in bacteria and then move the DNA into a eukaryote. Most eukaryotic vectors are shuttle vectors.