1-4.6.2 Yeast Artificial Chromosomes: YAC

•  First described in 1983 by Murray and Szostak, a yeast artificial chromosome has sequences to exist inside E. coli as a circular plasmid and contains sequences to maintain as linear nuclear chromosome in yeast.

•  As YAC vectors can accommodate 100-500 kb of insert DNA. The number of clones in a genomic library can be greatly reduced.

•  YAC vectors have following elements:

•  E. coli origin of replication

•  Yeast origin of replication

•  Elements of eukaryotic yeast chromosome (centromere and telomere region)

•  Selection markers for both the host.

•  YAC vector is initially propagated as circular plasmid inside bacterial host utilizing bacterial ori sequence. Circular plasmid is cut at specific site using restriction enzymes to generate a linear chromosome with two telomere sites at terminals. The linear chromosome is again digested at specific site with two arms with different selection marker. Genomic insert is then ligated into YAC vector using DNA ligase enzyme. The recombinant vectors are transformed into yeast cells and screened for the selection markers to obtain recombinant colonies.

•  Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs (yeast episomal plasmids), have advantageous over bacterial artificial chromosomes (BACs). They can be used to express eukaryotic proteins that require post-translational modification. However, YACs have been found to be less stable than BACs.

Some recombinant plasmids have the ability to incorporate multiple replication origins and other elements that allow them to be used in more than one species (for example, yeast or E. coli ).

Fig 1-4.6.2 : YAC vector

1-4.6.3 Mammalian Artificial Chromosomes: MAC

MACs or mammalian artificial chromosomes, like YACs, rely on the presence of centromeric and telomeric sequences and origin of DNA replication. They involve autonomous replication and segregation in mammalian cells, as opposed to random integration into chromosomes (as for other vectors). They can be modified for their use as expression systems of large genes, including not only the coding region but can contain control elements. Two principal procedures exist for the generation of MACs.

1)  In one method, telomere-directed fragmentation of natural chromosomes is used. For example, a human artificial chromosome (HAC) has been derived from chromosome 21 using this method.

2)  Another method involves de novo assembly of cloned centromeric, telomeric, and replication origins in vitro .

MAC vectors are difficult to assemble as compared to YAC vectors. Mammalian DNA has higher degree of repetition and larger centromere and telomere regions. Also the sequences necessary for chromosome replication in mammalian system are not well defined till now. MAC vectors have application in the field of gene therapy and eukaryotic protein expression and production.