2. CONTENTS
Introduction
Purpose and features
Yeast Artificial Chromosome Plasmids
Construction YAC
Homologous Recombination
Application
Comparison between YAC And BAC System
Research
3. Introduction
Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone
DNA sequences in yeast cells
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.
4. YAC is an artificially constructed chromosome that contains :
Centromere
Telomeres
Autonomous replicating sequence (ARS) element required for replication and
preservation of YAC in yeast cells
YACS behave like naturally existing chromosomes, provided that they are of the
proper size, showing comparable stability.
5. Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes.
Clone very large inserts of DNA :- 100 kb - 10 Mb.
Purpose & Features
One arm contains an autonomous replication sequence (ARS), a centromere (CEN)
and a selectable marker (trp1). The other arm contains a second selectable marker
(ura3).
Insertion of DNA into the cloning site inactivates a mutant expressed in the vector
DNA and red yeast colonies appear.
Transformants are identified as those red colonies which grow in a yeast cell that is
mutant for trp1 and ura3. This ensures that the cell has received an artificial chromosome
with both telomeres (because of complementation of the two mutants) and the artificial
chromosome contains insert DNA (because the cell is red).
6.
7. Yeast Integrating plasmids (YIp):
These plasmids lack an ORI and must be integrated directly into
the host chromosome via homologous recombination.
Yeast Replicating plasmids (YRp):
These vectors contain an Autonomously Replicating Sequence
(ARS) derived from the yeast chromosome. As the name suggests, these vectors can replicate
independently of the yeast chromosome; however, they tend to be unstable and may be lost
during budding.
The Four Main Types Of Yeast Plasmids Are:
8. Yeast Centromere plasmids (YCp):
These are considered low copy vectors and incorporate part of
an ARS along with part of a centromere sequence (CEN). These vectors replicate as
though they are small independent chromosomes and are thus typically found as a
single copy Unlike the ARS vectors, CEN vectors are stable without integration.
Yeast Episomal plasmids (YEp):
These are most similar to bacterial plasmids and are considered
“high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows
for 50+ copies to stably propogate per cell. The copy number of these vectors can
also be controlled if specific regulatable elements are included.
9. Construction
Ligation of selectable marker into plasmid vector: this allows for the differential
selection of colonies with, or without the marker gene An antibiotic resistance gene
allows the YAC vector to be amplified and selected for in E. coli by rescuing the
ability of mutant E. coli to synthesize leucine in the presence of the necessary
components within the growth medium. TRP1 and URA3 genes are other YAC vector
cloning site for foreign DNA is located within the SUP4 gene. This 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 colorless
colonies. Cloning of a foreign DNA fragment into the YAC causes insertional
inactivation of the gene, restoring the red color. Therefore, the colonies that contain the
foreign DNA fragment are red.
Ligation of necessary centromeric sequences for mitotic stability.
10.
11. Ligation of Autonomously Replicating Sequences (ARS) providing an origin of replication
to undergo mitotic replication Allows the plasmid to replicate extrachromosomally, but
renders the plasmid highly mitotically unstable, and easily lost without the centromeric
sequences.
Ligation of artificial telomeric sequences to convert circular plasmid into a linear piece of
DNA.
Insertion of DNA sequence (up to 1000kb).
Transformation yeast colony.
12. Homologous
Recombinantion
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
Firstly two YAC vectors arms and the exogenous segment (flanked
by desired sequences) are into the yeast cell
Then followed by recombination
Results in formation of desired stable YACS.
13. Recomentional Targeting 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.
14. 2
1 3 4
Application
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)
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
15. YAC Genomic Libraries
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
16.
17. Research…!!
Human artificial chromosome (H
for measuring chromosome inst
(CIN) and identification of gene
required for proper chromosom
transmission .
BY : - Natalaykouprina,Mikhail Liskovykh
Nikolai Petrov, Vladimir Larionov .
Journal name :- Experimental Cell Research
18. CHROMOSOMAL INSTABILITY CIN) is one of the characteristics of cancer inherent
for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss
of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN
remains unknown.
The development of a conceptually simple colony color sectoring assay that measures
yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate
of chromosome mis-segregation and also identified 937 yeast genes involved in this
process. Similarly, a human artificial chromosome (HAC)-based assay has been recently
developed and applied to quantify chromosome mis-segregation events in human cells.
Abstract
19. This assay allowed identification of novel human CIN genes in the library
of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1.
The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-
based libraries to identify the complete spectrum of CIN genes.
This will reveal new insights into mechanisms of chromosome segregation and
may expedite the development of novel therapeutic strategies to target the CIN
phenotype in cancer cells.
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THANKS !!
Thank you for
Your Patience ,
All The
BEST…!!
