Cloning into a Yeast Artificial Chromosome (YAC)
DNA cloning is a technique that allows the wholesale
production of a specific DNA sequence. DNA containing a gene of interest
is inserted into the purified DNA genome of
a self replicating element, which can be a plasmid, a virus or in this case,
a yeast artificial chromosome (YAC).
A YAC can be considered as a functional artificial chromosome (self replicating
element), since it includes three specific DNA sequences that enable it
to propagate from one cell to its offspring:
- TEL: The telomere which is located at each chromosome
end, protects the linear DNA from degradation by nucleases.
- CEN: The centromere which is the attachment site for
mitotic spindle fibers, "pulls" one copy of each duplicated chromosome
into each new daughter cell.
- ORI: Replication origin sequences which are specific DNA sequences
that allow the DNA replication machinery to assemble on the DNA and move
at the replication forks.
It also contains few other specific sequences like:
- A and B: selectable markers that allow the easy
isolation of yeast cells that have taken up the artificial chromosome.
- Recognition site for the two restriction
enzymes EcoRI and BamHI.
While DNA cloning into a plasmid allows the
insertion of DNA fragment of about 10,000 nucleotide base pairs, DNA cloning
into a YAC allows the insertion of DNA fragments up to 1,000,000 nucleotide
Why is it so important to be able to clone such large sequences? To map
the entire human genome (3x1,000,000,000 nucleotide base pairs) it would
require more than 100,000 plasmid clones. In principle, the human genome
could be represented in about 10,000 YAC clones.
Techniques for cloning genomic DNA into yeast artificial chromosomes
(YAC) make it possible to analyze very long DNA sequences like human genes.
Cloning human genomic DNA into a YAC:
- Genomic DNA is partially digested by the restriction enzyme EcoRI.
Very large DNA fragments are obtained.
- The YAC is digested by the two restriction enzymes EcoRI and BamHI.
- Those two elements recombine at the EcoRI sites and are covalently
linked by the DNA ligase.
- A recombinant YAC vector, a yeast artificial chromosome with genomic
DNA inserted, is produced. This vector can be used to infect yeast cells
and generated an unlimited number of copies.
Adapted from D.T. Burke, G.F. Carle, M.V. Olson. Science 236: 806-812,