Yeast Artificial Chromosomes (YACs)

2. 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.
Yeast artificial chromosome (YAC) is
a human-engineered DNA molecule
used to clone DNA sequences in
yeast cells

3. YEAST ARTIFICIAL CHROMOSOMES
 YAC is an artificially constructed chromosome that
contains a
 Centromere
 Telomeres
 Autonomous replicating sequence (ARS) element
required for replication and preservation of YAC
in yeast cells
 ARS elements are thought to act as replication origins
First described in 1983 by Murray and Szostak

4. YACs behave like naturally
existing chromosomes, provided
that they are of the proper size,
showing comparable stability.

5. Purpose:
 Cloning vehicles that propogate in eukaryotic cell
hosts as eukaryotic Chromosomes
 Clone very large inserts of DNA: 100 kb - 10 Mb
Features:
 YAC cloning vehicles are plasmids
 Final chimeric DNA is a linear DNA molecule with
telomeric ends: Artificial Chromosome

6. Relatively
small size
(approaximat
ely 12 kb)
Circular form
Amplified
in E. coli
Very large
size (several
hundreds of
kilobases)
Linear
Amplified
in yeast

7. Yeast Artificial Chromosomes
(pYACs) Plasmids
 Many different yeast artificial chromosomes
plasmids exist, such as 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
 Include yeast ARS1 with its associated CEN4
DNA sequence, as well as the URA3 selectable

8.  Yeast HIS3 is flanked by a telomere-like DNA
sequence 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.

9. Circular map of plasmid vector
pYAC3

10. Construction of Yeast Artificial
Chromosomes
Plasmid DNA purification
Treatment with restriction
enzymes
Ligation and yeast
transformation

11. CONSTRUCTION OF YAC
 A YAC is built using an initial circular plasmid
typically broken into two linear molecules
using restriction enzymes
 DNA ligase is then used to ligate a sequence or
gene of interest between the two linear molecules
 forming a single large linear piece of DNA

14.  This inserted gene compensates for a mutation in the yeast
host cell that causes the accumulation of red pigment
 The host cells are normally red, and those transformed with
YAC only, will form colourless colonies
 Cloning of a foreign DNA fragment into the YAC causes
insertional inactivation, restoring the red colour
 Therefore the colonies that contain the foreign DNA fragment
are red. The yeast artificial chromosome (YAC) vector is
capable of carrying a large DNA fragment (up to 2 Mb)
 Transformation efficiency is very low.

15. Homologous Recombination
 In recombinationally-targeted YAC
cloning, YACs are assembled in vivo, by
recombination, and not by ligation in
vitro
 Recombination takes place between a
target segment of the exogenous DNA,
and the YAC vector that contains
sequences homologous to these targets

16.  Firstly two YAC vectors arms and the
exogenous segment(flanked by desired
sequences) are transformed into the
yeast cell
 Then followed by recombination
 Results in formation of desired stable
YACs.

17. Figure.Recombinati
onal targeted
cloning with YAC
vectors. A yeast
strain is transformed
with a mixture of the
two YAC vector
arms and large
fragments of DNA.
Recombination in
vivo results in the
formation of a
specific YAC clone.
The two YAC vector
arms are derived
from linearized
plasmids that
contain targeting
segments that are
homologous to the
termini of the DNA
segment that is to
be cloned.

18. Existing YAC clones can be
modified by homologous
recombination in yeast
•‘Retrofitting’
Modifying YACs by Homologous
Recombination

19. Applications of YACs include generating whole DNA
libraries of the genomes of higher organisms
to identifying essential mammalian chromosomal
sequences necessary for the future construction of
specialized mammalian artificial chromosomes
(MACs)
Use of Yeast Artificial
Chromosomes

20. Another major application of YACs is in the study of
regulation of gene expression by cis-acting,
controlling DNA elements
That are present either upstream or downstream of
large eukaryotic genes, after the transfer of these
YACs from yeast to mammalian cells

21.  It is possible to construct YACs with
megabase-long inserts using the precise
homologous recombination
 original DNA sequence of a eukaryotic
genome fragment more than 2Mb in size
can be maintained in a single YAC
vector
YAC Genomic Libraries

22. COMPARISON BETWEEN YAC
AND BAC SYSTEMS
FEATURE YAC BAC
Configuration Linear
Circular
Host Yeast Bacteria
Copy Number / Cell 1 1-2
Cloning Capacity Unlimited None to low
Chimerism Up to 40%
Insert Stability Unstable
Stable