Yeast Artificial Chromosome also known as YAC is an artificial vector used in molecular biology for genetic recombination, gene cloning and biotechnology.

1. YEAST
ARTIFICIAL
CHROMOS
OME
SAJIN DANGOL

2. INTRODUCTION
The plasmids can accommodate about 10kb foreign DNA, Phage can
accommodate about 20kb DNA insert, Cosmid can accommodate about 40kb
long four DNA segments.
David and co workers in 1987 developed Yeast Artificial Chromosomes (YAC)
where DNA segment of several thousand base pairs (about 1 Mb) can be cloned.
It has also been used for physical mapping of human chromosome in “Human
Genome Project”.

3. Yeast artificial chromosomes (YACs) are plasmid shuttle vectors capable of
replicating and being selected in common bacterial hosts such as Escherichia
coli, as well as in the budding yeast Saccharomyces cerevisiae.
They are of relatively small size (approximately 12 kb) and of circular form when
they are amplified or manipulated in E. coli, but are rendered linear and of very
large size, i.e. several hundreds of kilobases (kb), when introduced as cloning
vectors in yeast.

4. In this linear form, these specialized vectors contain all three cis-acting structural
elements essential for behaving like natural yeast chromosomes:
1. an autonomously replicating sequence (ARS) necessary for replication;
2. a centromere (CEN) for segregation at cell division; and
3. two telomeres (TEL) for maintenance.
In addition, their capacity to accept large DNA inserts enables them to reach the
minimum size (150 kb) required for chromosome-like stability and for fidelity of
transmission in yeast cells.

5. Overview of Yeast Artificial
Chromosomes (pYACs) Plasmids
Many different yeast artificial chromosomes exist as ongoing refinements of the
initial pYAC3 and pYAC4 plasmids.
The basic structural features of YACs were developed from the yeast centromere
shuttle-plasmids YCp series.
These are composed of double-stranded circular DNA sequences carrying the b-
lactamase gene bla and the bacterial pMB1 origin of replication, thus conferring
resistance to ampicillin and the ability to replicate in bacteria, respectively.

6. They also include yeast ARS1 with its associated CEN4 DNA sequence, as well as the
URA3 selectable marker.
On this basic scaffold plasmid the yeast HIS3 is cloned, flanked by a telomere-like
DNA sequence derived from the termini of the Tetrahymena pyriformis macronuclear
ribosomal DNA, which allows the formation of functional telomeres in yeast, that are
adjacent to two recognition sites for the BamHI restriction enzyme.
Most of these YACs also contain the cloning site in the middle of the SUP4 suppressor
of an ochre allele of a tyrosine transfer RNA (tRNA) gene; this enables restoration of the
normal white colour phenotype in otherwise red ade1 and/or ade2 nonsense mutants.

9. Construction of Yeast Artificial
Chromosomes
After plasmid DNA purification, two distinct digestions are performed:
the first with BamHI that cuts twice adjacent to the two telomeric DNA sequences
flanking the HIS3 gene, which therefore is excised from the plasmid and lost.
 This first digestion generates a long linear fragment carrying telomeric sequences at
each end.
The second digestion consists of the opening of the cloning site within the SUP4 gene.
As a result of this second digestion, two linear fragments are produced as left and right
arms of the future linear YAC.

10. The selective markers are thus separated: TRP1 adjacent to ARS1 and CEN4 on
the left arm and URA3 on the right arm.
Large DNA fragments with ends compatible to the cloning site, obtained from
the desired genome source by digestion with an appropriate restriction
endonuclease, are ligated with phosphatase-treated YAC arms, to create a single
yeast-transforming DNA molecule.
Primary transformants can be selected for complementation of the ura3
mutation in the host, and successively for complementation of the host trp1
mutation, thereby ensuring the presence of both chromosomal arms.
Transformant colonies containing the exogenous DNA insert within the SUP4
gene are detected by their red colour, due to the inactivation of the suppressor
activity and the consequent accumulation of a red metabolic precursor in ade host
cells.

14. Advantages of Yeast Artificial
Chromosomes
•Yeast artificial chromosomes (YACs) provide the largest insert capacity of any cloning system.
•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.
•A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unstable,
underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in
YAC clones.
•It is possible to reintroduce YACs intact into mammalian cells where the introduced mammalian
genes are expressed and used to study the functions of genes in the context of flanking
sequences.

15. Uses of Yeast Artificial
Chromosomes
•Yeast artificial chromosomes (YACs) were originally constructed in order to
study chromosome behavior in mitosis and meiosis without the complications of
manipulating and destabilizing native chromosomes.
•YACs representing contiguous stretches of genomic DNA (YAC contigs) have
provided a physical map framework for the human, mouse, and even Arabidopsis
genomes.
•YACs are extremely popular for those trying to analyze entire genomes.

16. Limitations
A problem encountered in constructing and using YAC libraries is that they typically contain clones that are
chimeric, i.e., contain DNA in a single clone from different locations in the genome.
YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of the cloned DNA. As
a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA have
occurred.
The efficiency of cloning is low (about 1000 clones are obtained per microgram of vector and insert DNA).
YACs have been found to be less stable than BACs.
The yield of YAC DNA isolated from a yeast clone containing a YAC is quite low.
The YAC DNA is only a few percents of the total DNA in the recombinant yeast cell. It is difficult to obtain
even 1 μg of YAC DNA.