Yeast Artificial Chromosomes (YACs)

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

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

YACs behave like naturally

existing chromosomes, provided

that they are of the proper size,

showing comparable stability.

Purpose:

Cloning vehicles that propogate in eukaryotic cellhosts 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 withtelomeric ends: Artificial Chromosome

Relatively small size

(approaximately 12 kb)

Circular form

Amplified in E. coli

Very large size (several hundreds of kilobases)

Linear

Amplified in yeast

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

marker.

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.

Circular map of plasmid vector

pYAC3

Construction of Yeast ArtificialChromosomes

Plasmid DNA purification

Treatment with restriction enzymes

Ligation and yeast transformation

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

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.

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

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.

Figure.Recombinational targetedcloning with YACvectors. A yeaststrain is transformedwith a mixture of thetwo YAC vectorarms and largefragments of DNA.Recombination invivo results in theformation of aspecific YAC clone.The two YAC vectorarms are derivedfrom linearizedplasmids thatcontain targetingsegments that arehomologous to thetermini of the DNAsegment that is tobe cloned.

Existing YAC clones can bemodified by homologousrecombination in yeast

• ‘Retrofitting’

Modifying YACs by Homologous

Recombination

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

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

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

COMPARISON BETWEEN YAC

AND BAC SYSTEMS

FEATURE YAC BAC

Configuration LinearCircular

Host Yeast Bacteria

Copy Number / Cell 1 1-2

Cloning Capacity Unlimited None to low

Chimerism Up to 40%

Insert Stability UnstableStable