between thecossites is 52 kb. Consequently cosmid vectors have been constructed
that incorporate thecossites from phageland also the essential features of a plasmid,
such as the plasmid origin of replication, a gene for drug resistance, and several
unique restriction sites for insertion of the DNA to be cloned. When a cosmid
preparation is linearised by restriction digestion, and ligated to DNA for cloning, the
products will include concatamers of alternating cosmid vector and insert. Thus the
only requirement for a length of DNA to be packaged into viral heads is that it should
containcossites spaced the correct distance apart; in practice this spacing can range
between 37 and 52 kb. Such DNA can be packagedin vitroif phage head precursors,
tails and packaging proteins are provided. Since the cosmid is very small, inserts of
about 40 kb in length will be most readily packaged. Once inside the cell, the DNA
recircularises through itscossites, and from then onwards behaves exactly like a
plasmid.6.3.5 Large insert capacity vectors
The advantage of vectors that accept larger fragments of DNA than phagelor cosmids is
that fewer clones need to be screened when searching for the foreign DNA of interest.
They have also had an enormous impact in the mapping of the genomes of organisms
such as the mouse and are used extensively in the human genome mapping project
(Section 6.9.3). Recent developments have allowed the production of large insert capacity
vectorsbased on human artificial chromosomes, bacterial artificial chromosomes (BACs),
mammalian artificial chromosomes (MACs) and on the virus P1 (PACs), P1 artificial
chromosomes. However, perhaps the most significant development are vectors based on
yeast artificial chromosomes (YACs).6.3.6 Yeast artificial chromosome (YAC) vectors
Yeast artificial chromosomes (YACs) are linear molecules composed of a centromere,
telomere and a replication origin termed an ARS element (autonomous replicating
sequence). The YAC is digested with restriction enzymes at the SUP4 site (a suppressor
tRNA gene marker) andBamHI sites separating the telomere sequences (Fig. 6.24).
This produces two arms and the foreign genomic DNA is ligated to produce a
functional YAC construct. YACs are replicated in yeast cells; however, the external
cell wall of the yeast needs to be removed to leave a spheroplast. These are osmotically
unstable and also need to be embedded in a solid matrix such as agar. Once the yeast
cells are transformed only correctly constructed YACs with associated selectable
markers are replicated in the yeast strains. DNA fragments with repeat sequences
are sometimes difficult to clone in bacterial-based vectors but may be successfully
cloned in YAC systems. The main advantage of YAC-based vectors, however, is the
ability to clone very large fragments of DNA. Thus the stable maintenance and
replication of foreign DNA fragments of up to 2000 kb have been carried out in
YAC vectors and they are the main vector of choice in the various genome mapping
and sequencing projects (Section 6.9).220 Recombinant DNA and genetic analysis